MA1-514

antibody from Invitrogen Antibodies

Targeting: AHR bHLHe76

Provider product page for MA1-514

Western blot

Immunocytochemistry

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [45]
Comments [0]
Validations
Western blot [4]
Immunocytochemistry [4]
Immunohistochemistry [2]
Other assay [24]

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Product number: MA1-514 - Provider product page
Provider: Invitrogen Antibodies
Product name: AHR Monoclonal Antibody (RPT1)
Antibody type: Monoclonal
Antigen: Synthetic peptide
Description: MA1-514 detects the aryl hydrocarbon receptor (AhR) from human, mouse, and rat cells.
Antibody clone number: RPT1
Concentration: Conc. Not Determined

AHR protein structure - MA1-514 shown in red.

Supportive validation

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Experimental details: Western blot analysis of Aryl Hydrocarbon Receptor was performed by loading 20 µg of HepG2, U2OS, and K562 whole cell lysates and 5uL of PageRuler Plus Prestained Protein Ladder (Product # 26619) per well onto a 4-20% Tris-Glycine polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane using the G2 Blotter (Product # 62288), and blocked with 5% milk in TBST for 1 hour at room temperature. Aryl Hydrocarbon Receptor was detected at ~96 kD using an AhR monoclonal antibody (Product # MA1-514) at a dilution of 1:1000 in 5% milk in TBST overnight at 4C on a rocking platform, followed by a goat anti-mouse IgG-HRP secondary antibody (Product # 31430) at a dilution of 1:10,000 for at least 30 minutes at room temperature. Chemiluminescent detection was performed using SuperSignal West Pico substrate (Product # 34080).

Supportive validation

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Experimental details: Knockout of AHR was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR980382_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of AHR was performed by loading 60 µg of A-375 wild type (Lane 1), A-375 Cas9 (Lane 2) andA-375 AHR KO (Lane 3) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-AHR Monoclonal Antibody (RPT1) (Product # MA1-514, 1:2000 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:8000 dilution) using the iBright™ FL 1500 (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to AHR. Uncharacterized band was observed in all the samples at ~32 kDa.

Supportive validation

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Experimental details: Knockdown of AHR was achieved by transfecting A-375 with AHR specific siRNAs (Silencer® select Product # s1198). Western blot analysis (Fig. a) was performed using whole cell extracts from the AHR knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with AHR Monoclonal Antibody (Product # MA1-514, 1:1000 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to AHR.

Supportive validation

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Experimental details: Western blot was performed using Anti-AHR Monoclonal Antibody (Product # MA1-514) and a 100 kDa band corresponding to AHR was observed in the cell lines tested except U-87 MG, Jurkat and MOLT-4 which are reported to be negative. Whole cell extracts (30 µg lysate) of A-375 (Lane 1), U-87 MG (Lane 2), T98G (Lane 3), Jurkat (Lane 4) and MOLT-4 (Lane 5 were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of Aryl Hydrocarbon Receptor in HeLa cells. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with a Aryl Hydrocarbon Receptor monoclonal antibody (Product # MA1-514) at a dilution of 1:200 overnight at 4 C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35503). Aryl Hydrocarbon Receptor staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Images were taken at 60X magnification.

Supportive validation

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Experimental details: Immunofluorescent analysis of Aryl Hydrocarbon Receptor in MCF-7 cells. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with a Aryl Hydrocarbon Receptor monoclonal antibody (Product # MA1-514) at a dilution of 1:100 overnight at 4 C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35503). Aryl Hydrocarbon Receptor staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Images were taken at 60X magnification.

Supportive validation

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Experimental details: Immunofluorescent analysis of Aryl Hydrocarbon Receptor in NIH-3T3 cells. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with a Aryl Hydrocarbon Receptor monoclonal antibody (Product # MA1-514) at a dilution of 1:200 overnight at 4 C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35503). Aryl Hydrocarbon Receptor staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Images were taken at 60X magnification.

Supportive validation

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Experimental details: Immunofluorescence analysis of AHR was performed using 70% confluent log phase A-375 cells. The cells were fixed with 4% Paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 10 minutes at room temperature. The cells were labeled with AHR Monoclonal Antibody (RPT1) (Product # MA1-514) at 1:200 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, Alexa Fluor 488 (Product # A28175), (1:2000 dilution) for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing Cytoplasmic localization. Panel e represents MOLT-4 cells having no expression of AHR. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

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Experimental details: Immunohistochemistry was performed on cancer biopsies of deparaffinized Human breast carcinoma tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature and probed with a Aryl Hydrocarbon Receptor monoclonal antibody (Product # MA1-514) at a dilution of 1:100 or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry was performed on normal biopsies of deparaffinized Human skeletal muscle tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature and probed with a Aryl Hydrocarbon Receptor monoclonal antibody (Product # MA1-514) at a dilution of 1:20 or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Supportive validation

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1. AhR is expressed in mouse testis and it does not significantly affect testis weight or size. ( a ) Testes from 24 dpp AhR +/+ and AhR -/- mice were extracted, processed histologically and stained with haematoxylin-eosin. ( b , c ) Testes weight ( b ) and size ( c ) were determined in mice of each genotype at the indicated times after birth. Testis size was calculated using the formula [length x width 2 x 0.4]. ( d ) AhR mRNA expression was quantified by RT-qPCR in AhR +/+ testes at the indicated ages, using total RNA and the specific primers indicated in Material and methods. RNA isolated from adult AhR -/- testes was used as negative control. RT-qPCR data were normalized by the expression of Gapdh and represented as 2 -DeltaDeltaCt . ( e ) AhR protein expression was determined by immunoblotting in AhR +/+ testes at the indicated ages using total protein extracts. The expression of beta-actin was used to normalize protein loading. The graph represents the profile of AhR protein expression over time. ( f ) Immunofluorescence analysis of AhR expression in AhR +/+ and AhR -/- mouse testes at 10, 12 and 14 dpp. Arrowheads mark the presence of AhR (red fluorescence) in the seminiferous tubules. Testes extracted from AhR -/- mice were used as negative controls. DAPI was used to stain cell nuclei. Details of the micrographs are shown in the insets. Four biological replicates and two experimental duplicates were done for panels ( d,e ). At least four individual mice of each

Supportive validation

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Experimental details: Figure 7. AhR deficiency reduces the number of follicles in the ovary. ( a ) Ovaries were extracted from 5 and 15 dpp, and adult (six to eight weeks) AhR +/+ mice and used to obtain total RNA. AhR mRNA expression was quantified by RT-qPCR as indicated in Material and methods. Gene expression was normalized by Gapdh and represented as 2 -DeltaDeltaCt . Ovaries from adult AhR -/- mice were used as negative control. ( b ) Ovaries from AhR +/+ mice at the ages indicated above were used to prepare total protein extracts. AhR protein expression was determined by immunoblotting as indicated in Material and methods. Ovaries from adult AhR -/- mice were used as negative control. ( c ) AhR +/+ and AhR -/- ovaries were processed for immunofluorescence and stained with an AhR-specific antibody. Alexa-633-labelled secondary antibody was used to detect AhR expression. DAPI staining was added to label cell nuclei. Arrowheads mark AhR expression. ( d , e ) The number of follicles was quantified in 5 ( d ) and 15 dpp ( e ) AhR +/+ and AhR -/- ovaries. ( f ) Haematoxylin-eosin staining was performed to analyse the follicles present in adult AhR +/+ and AhR -/- ovaries. ( g,h ) Oocytes were extracted from the ovary ( g ) or the ampulae of the oviduct ( h ) of adult mice of both genotypes. At least five biological replicates were analysed for each genotype. Duplicate or triplicate experimental determinations were done. Data in panels ( a,d,e,g,h ) are shown as means +- s.d. ** p < 0.01, *** p

Supportive validation

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Experimental details: Figure 9. Adult AhR- mice have reduced numbers of MVH- and Miwi-positive ovarian follicles. ( a ) Ovaries from adult (five to six weeks) AhR +/+ and AhR -/- mice were extracted and processed for the detection of MVH, Mili and Miwi by immunofluorescence. An Alexa-633-labelled secondary antibody has been used. ( b ) The number of positive follicles was quantified for each individual ovary of each mouse genotype. Ovaries from at least four AhR +/+ and AhR -/- mice were used, and immunofluorescences were done in triplicate. Data in panel ( b ) are shown as mean +- s.d. * p < 0.05, ** p < 0.01. Adult mice were 12-13 weeks of age. Scale bar, 50 um.

Supportive validation

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Experimental details: Fig. 1 TCDD, beta-NF and ITE induce AhR activation in NHEK, (A) Primary normal human epidermal keratinocytes (NHEKs) were co-transfected with XRE4-SV40-luciferase and renilla-luciferase constructs and treated with vehicle, TCDD, beta-NF or ITE as indicated for 48 h. XRE-luciferase activity was measured and firefly:renilla luciferase ratio was normalised to vehicle. Data shown are mean +- sem, n = 9 (triplicate wells from 3 donors). Analysis of trend comparing vehicle to ligand, *** P < 0.0004. (B) Primary NHEKs were treated every 48 h for 8 days (on days 0, 2, 4 and 6) with vehicle or ligand as indicated. Samples were lysed and Western blotting performed. (C) Densitometry was carried out on blots probed with antibodies against AhR and CYP1A1 and normalised to beta-actin. Densitometry represents mean +- sem from 3 donors; graph shows results of one-way ANOVA. Effects of TCDD on AhR: one-way ANOVA, ** P = 0.007, analysis of trend P = 0.02. Effects of beta-NF on AhR: one-way ANOVA, * P = 0.02, analysis of trend, NS. Effects of ITE on AhR: one-way ANOVA, *** P = 0.0002, analysis of trend P < 0.02.

Supportive validation

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Experimental details: Fig. 6 TCDD-induced AhR activation is inhibited by alpha-NF in primary NHEKs. (A) Primary NHEKs were co-transfected with XRE4-SV40-luciferase and renilla-luciferase control constructs and treated with vehicle or TCDD and/or alpha-NF as indicated for 48 h. Luciferase activity was measured and the firefly:renilla luciferase ratio was normalised to vehicle. Data shown are mean (+-sem), n = 9 (triplicate wells from 3 donors). Two-way ANOVA: post hoc tests comparing vehicle to TCDD +- alpha-NF, Dunnett's: **/*, analysis of trend: *** P < 0.0001. (B) Primary NHEKs were treated with vehicle, TCDD and/or alpha-NF every 48 h as indicated for 8 days. Cells were lysed and proteins separated by Western blotting. Blots were probed with AhR and CYP1A1 antibodies with beta-actin as loading control. Western blot is representative of duplicate blots from 3 donors. (C) Densitometry was performed on blots from 3 donors probed with antibodies against AhR (black bars) and CYP1A1 (white bars) and normalised to beta-actin. Two-way ANOVA and analysis of trend: effects of TCDD on AhR, * P = 0.02. Densitometry is represents mean (+-sem) from 3 donors.

Supportive validation

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Experimental details: Figure 3 Bioinformatics analysis points to the involvement of Ahr in miR-132/212 induction following outlet obstruction. Transcription factor binding site (TFBS) analysis identified significant enrichment of 93 transcription factor binding motifs in promoters of differentially expressed genes at 10 days of obstruction (blue circle in A ). These were cross-referenced against putative transcription factor binding sites in the miR-132/212 promoter (red circle in A ), yielding a list of putative mediators of miR-132/212 induction in outlet obstruction ( B ). Hits with low expression levels and those with raw p-values exceeding 0.01 are shown in thin lettering. Panel C shows phosphorylation of CREB (S133) in bladders from sham-operated rats and following various time of obstruction. Panel D shows immunofluorescence labeling (green) of the dioxin receptor Ahr in bladders from sham-operated and obstructed rats. Dotted lines indicate the outer surface of the urinary bladder. The scale bar to the left applies to all images and represents 100 mum. Panels E through G show miR-132 expression in human bladder smooth muscle cells stimulated with vehicle (control) and various pharmacological substances in vitro. TCDD: 2,3,7,8-tetrachlorodibenzo-p-dioxin; FCS: fetal calf serum; PMA: Phorbol 12-myristate 13-acetate. Further details are given in Materials and Methods .

Supportive validation

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Experimental details: Supplementary Fig. I TCDD resulted in time and dose dependent AhR degradation and CYP1A1 induction between 2 and 8 days. (A) Primary NHEKs were treated for up to 8 days with vehicle, 5 or 10 nM TCDD as indicated. Samples were lysed and Western blotting performed. Densitometry was carried out on blots probed with antibodies against (B.i) AhR and (B.ii) CYP1A1 and normalised to beta-actin. Densitometry represents mean +- sem from 3 donors. Two-way ANOVA: analysis of trend on effects of TCDD on AhR: * P = 0.02. Effects of time and TCDD on CYP1A1 induction, analysis of trend: ** P < 0.002.

Supportive validation

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Experimental details: Figure 5 AhR nuclear localization in LNCaP and HepG2 cells. Western blot analysis was performed on nuclear protein extracts obtained from both prostate (LNCaP) and hepatic (HepG2) cancer cellular models treated with 10 uM beta-HCH following or not a 2 h pretreatment with 150 nM CH223191. The presence of AhR in the nuclear fraction is more noticeable in the samples corresponding to beta-HCH stimulated cells with respect to control untreated cells. The amount of nuclear AhR is similar to control if cells are treated with 10 uM beta-HCH in the presence of the AhR antagonist CH223191. Lamin A was used as a specific nuclear marker and normalization protein. The experiment was repeated three times and a representative blot is reported.

Supportive validation

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Experimental details: Figure 6 Cellular distribution of AhR followed by immunofluorescence in LNCaP ( A ) and HepG2 ( B ) cells. CTR: Control untreated cells; beta-HCH: Cells after 4 h of 10 uM beta-HCH stimulation; beta-HCH + CH223191: Cells after 2 h pre-incubation with 150 nM CH223191 followed by 4 h of 10 uM beta-HCH stimulation.

Supportive validation

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Experimental details: Figure 7 AhR proteasomal degradation is induced by beta-HCH in both LNCaP and HepG2 cells. ( A ) Total protein extracts were subjected to Western blot analysis. Samples obtained from cells treated with 10 uM beta-HCH alone exhibit a weaker signal compared to the samples from cells treated with both the inhibitors. The 2 h pretreatment step with 2 uM MG132 or 150 nM CH22131 is able to maintain AhR at the same levels as the control. ( B ) Quantification of protein levels by densitometric analysis. Values for each sample were normalized against the beta-actin level present in the same sample and taken as a housekeeping control. The experiment was repeated three times and a representative blot is reported. Statistically significant differences (** p < 0.01) are marked with asterisks.

Supportive validation

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Experimental details: Figure 2 Aryl hydrocarbon receptor (AHR) expression in C57BL/6 mouse liver. The antibody used against AHR was effective (coffee stain). Likewise, the samples were also stained with hematoxylin (blue) to contrast the cell nuclei Scale bar = 100 mum.

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Experimental details: Figure 3 AHR expression increases in the old hippocampus. We determined AHR expression by immunostaining and found that AHR expression increases in the elderly. The higher expression was found in non-neuronal cells. Representative images ( A ), total quantification ( B ), and AHR staining quantification in neurons and non-neuronal cells from the elderly ( C ). Arrows show the AHR staining. Values are mean +- SEM; Statistics were performed by unpaired t-test. Young (n = 7): 20-30 years old. Elder ( n = 6): >60 years old. Scale bar = 50 mum.

Supportive validation

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Experimental details: Figure 4 AHR and GFAP colocalization in post-mortem tissue. We determined AHR and GFAP expression by immunostaining and found AHR in astrocytes, apparently as microvesicles. Representative images from a 94 year-old female donor ( A , scale bar = 50 mum) and a 100 year-old Alzheimer''s disease (AD) female donor ( B , scale bar = 20 mum). The arrows show the AHR staining in astrocytes. The arrowheads show AHR with the appearance of microvesicles. We analyzed samples from three donors.

Supportive validation

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Experimental details: Figure 2 Biomarker immunohistochemical expression. ( a ) Membranous high expression of N-cadherin on tumour cells of PTC infiltrating the airway (PTC-A) compared with ( b ) an intrathyroid PTC (PTC-B); ( c ) PTC-A presented a low expression of E-cadherin and ( d ) PTC-B (follicular variant) with retained expression of this molecule; ( e ) PTC-A showing a high expression of AhR in contrast with ( f ), which shows low expression of the same immunomarker in a PTC-B; ( g ) CD147 high expression of PTC-B and ( h ) absent expression in PTC-A. Original magnification: ( a - d , h ) 200x; ( e - g ) 400x.

Supportive validation

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Experimental details: Figure 4 Cellular distribution of AhR followed by immunofluorescence in LNCaP cells. As displayed in the figures, receptor is only present in nuclei of cells treated with beta-HCH alone, but not in presence of CH223191 nor TOBC. Selected images are representative of three independent experiments and were captured under same acquisition parameters CTR: control untreated cells; beta-HCH: cells subjected to a 4 h stimulation with 10 uM beta-HCH; beta-HCH + CH223191: cells after 2 h pre-incubation with 150 nM CH223191, followed by 4 h of 10 uM beta-HCH stimulation; TOBC+ beta-HCH: cells pretreated 3 h with 1 mg/mL TOBC, and then for 4 h with 10 uM beta-HCH. CH223191 was used as a positive control for AhR inhibition.

Supportive validation

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Experimental details: Figure 5 Anakinra activates a xenobiotic sensing pathway via IDO1. ( A ) H1L1 cells were treated with different doses of kynurenine and anakinra for 6 hours and assessed for luciferase assay. ( B ) Representative immunoblots of IL1RA, HSP90, AIP, and AhR and ( C ) ARNT and AhR in cell lysates in which AhR was immunoprecipitated from Il1r1 -/- MEF cells treated with 10 mug/mL anakinra or 10 muM FICZ. In B and C , data are representative of 1 out of 2 independent experiments and the relative densitometric analysis are reported. ( D and E ) Il1r1 -/- MEF cells were treated with 10 mug/mL anakinra for 2 and 8 hours and analyzed for gene expression by a custom QuantiGene plex gene expression assay. Fold changes are reported as heatmap for 2 and 8 hours ( D ) and histograms for 8 hours ( E ) (data are representative of 1 out of 2 independent experiments). ( F ) Il1r1 -/- MEF cells were treated with 10 mug/mL anakinra and analyzed for mRNA expression of selected genes by RT-PCR ( n = 3 independent samples) and protein expression of IDO1 and SOCS3 by immunoblotting (representative experiment). The relative densitometric analysis is reported. ( G ) Il1r1 -/- MEF cells were treated with either 10 mug/mL anakinra for different times or 10 ng/mL IFN-gamma as positive control for 48 hours and assessed for kynurenine production by ELISA. ( H ) Il1r1 -/- MEF cells were treated with 10 mug/mL anakinra in the presence or absence of 10 muM epacadostat and assessed for kynurenine production by

Supportive validation

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Experimental details: Figure 6 OPN preserves the gut microbiome by inducing AMPs expression in IECs, which maintains intestinal barrier function and protects from ALD. WT and Opn KI IEC mice were fed control or ethanol diet for 6 weeks to provoke ALD. mRNA expression of Reg3beta , Reg3gamma , and Il33 in IECs isolated from jejunum. Data were normalized with Gapdh as housekeeping gene and FC was calculated against WT control (n = 6 [3 males + 3 females]/group) ( A ). Western blot of REG3G and total AHR in IECs isolated from jejunum (n = 3 males/group) ( B ). Western blot of STAT3 and pSTAT3 in IECs isolated from jejunum (n = 3 males/group) ( C ). Levels of Trp, its metabolites kynureine and indole metabolites (3-indoleacetic acid + 3-indoleacrylic acid + 3-indolepropionic acid + 3-indoxyl sulfate + indole + indole-3-lactic acid + tryptamine + tryptophol) in portal serum (n = 3 males/group) ( D-E ). Levels of SCFAs (acetic acid + propionic acid + butyric acid) in portal serum (n = 3 males/group) ( F-G ). mRNA expression of Ahr , Occludin , Claudin3 , JamA , and Jam4 in IECs from jejunum. Data were normalized with Gapdh as housekeeping gene and FC was calculated against WT control (n = 6 [3 males +3 females]/group) ( H ). Data are expressed as mean +- standard error of the mean (SEM). * P < .05; ** P < .01; and *** P < .001 vs control; # P < .01 vs WT control; ^ P < .05; ^^ P < .01; and ^^^ P < .01 vs WT ethanol. WT and Opn DeltaIEC mice were fed control or ethanol diet for 6 weeks to provoke ALD. mR

Supportive validation

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Experimental details: Oral administration of mOPN induces AMPs in IECs, which preserve the gut microbiota and the intestinal barrier function. WT mice were fed control or ethanol diet for 6 weeks in the presence or absence of mOPN. mRNA expression of Reg3beta , Reg3gamma , and Il33 in IECs from jejunum. Data were normalized with Gapdh as housekeeping gene, and FC was calculated against without mOPN control (n = 4/group) ( A ). REG3G protein expression in IECs from jejunum (n = 3/group) ( B ). mRNA expression of Ahr , Occludin , Claudin3 , JamA , and Jam4 in IECs from jejunum. Data were normalized with Gapdh as housekeeping gene, and FC was calculated against without mOPN control (n = 4/group) ( C ). Western blot of total, cytosolic and nuclear AHR in IECs from jejunum (n = 3/group) ( D ). Levels of Trp, its metabolites kynureine and indole metabolites (3-indoleacrylic acid + 3-indoleacrylic acid + 3-indolepropionic acid + 3-indoxyl sulfate + indole + indole-3-lactic acid + tryptamine + tryptophol) in portal serum (n = 3/group) ( E-F ). Level of SCFAs (acetic acid + propionic acid + butyric acid) in portal serum (n = 4/group) ( G-H ). Data are expressed as mean +- standard error of the mean. * P