40-3700

antibody from Invitrogen Antibodies

Targeting: TXNIP ARRDC6, EST01027, HHCPA78, THIF, VDUP1

Provider product page for 40-3700

Western blot

Immunoprecipitation

Other assay

Antibody data

Antibody Data
Antigen structure
References [20]
Comments [0]
Validations
Western blot [1]
Other assay [22]

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Product number: 40-3700 - Provider product page
Provider: Invitrogen Antibodies
Product name: TXNIP Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Reactivity: Human, Bovine
Host: Rabbit
Isotype: IgG
Vial size: 100 µg
Concentration: 0.25 mg/mL
Storage: -20°C

TXNIP protein structure - 40-3700 shown in red.

Supportive validation

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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: NULL

Supportive validation

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

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
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: Fig. 2. A : verification of gene array result by qRT-PCR. Cells were exposed to either 250 muM or 5 mM Cr. While 250 muM Cr did not change thioredoxin-interacting protein (Txnip) mRNA (107 +- 3.7; P = 0.449), saturating Cr caused a rise in Txnip mRNA levels in agreement to the gene array observation ( n = 3 from 3 independent experiments; 161 +- 2; P = 0.0016). Moreover, the induction of Txnip expression was specific for Cr and did not occur at 5 mM taurine ( n = 3; 106; P = 0.5481). B : Txnip protein levels were elevated by 5 mM Cr, at 3 h ( n = 3/treatment; 145 +- 11; P = 0.0139), whereas neither 250 muM Cr nor taurine changed protein expression. ns, Nonsignificant P > 0.05, * P < 0.05, and ** P < 0.005.

Supportive validation

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Experimental details: Figure 1 TXNIP is highly expressed in DTC cell lines and low or undetectable in ATC cell lines, and glucose uptake is inversely proportional to TXNIP expression levels. (A) HTh74 cells were transduced with lentivirus expressing a PPARgamma-specific shRNA or scrambled control, and stable pools were generated under antibiotic selection. Western blot analysis of nuclear lysates (top immunoblot) reveals PPARgamma expression levels in the transduced cells. Using Western blot analysis of whole cell lysates, TXNIP and beta-actin (loading control) were detected using specific antibodies. Nonspecific band is indicated by ""ns"". (B) Western blot analysis for TXNIP, Trx-1, and beta-actin (loading control) were performed on whole cell lysates prepared from a panel of DTC and ATC cell lines grown under standard conditions. (C) Glucose uptake assays were performed. Each condition was performed in triplicate per experiment, and each experiment was performed at least three times. Nonspecific glucose uptake as determined by parallel treatment of a subset with cytochalasin B was subtracted from measurements. Data from all experiments were combined, and glucose uptake from each cell line was normalized to levels of HTh74 (average set at 1). Normalized averages are plotted, and error bars indicate SEM. ***p

Supportive validation

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Experimental details: Figure 2 TXNIP is expressed in primary DTC tumors but is low or undetectable in ATC tumors. Immunohistochemistry was performed using a TXNIP-specific primary antibody or normal mouse IgG control and a horseradish peroxidase tagged secondary antibody on a panel of 13 PTC and 8 ATC patient specimens. Two representative PTC (A-B) and one ATC (C) specimen stains are shown at 20X and 60X power. Positive staining is indicated by brown coloring, though staining of colloid is nonspecific. Staining was quantitated by a pathologist (S. B. S.), and intensity of staining was scored from absent (0) to high to (3+), followed by percent of tumor positivity.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 TXNIP overexpression in ATC HTh74 and T238 cells attenuates glucose uptake. HTh74 and T238 cells were transduced with retrovirus encoding human TXNIP or vector control as well as a selectable antibiotic resistance marker, and stable pools were generated under antibiotic selection. Western blot analysis of whole cell lysates with TXNIP- and beta-actin-specific antibodies is shown for HTh74 (A) and T238 (B) . Glucose uptake assays were performed as described in Figure 1 using the HTh74 stable cell lines (C) and T238 stable cell lines (D) . Data from all experiments were combined, and glucose uptake from each cell line was normalized to vector control levels (average set at 1). **p = 0.001, ***p

Supportive validation

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Experimental details: Figure 4 TXNIP overexpression inhibits in vitro growth of ATC HTh74 cells. Viable cell proliferation assays were performed for stable HTh74 (A) and T238 (B) cell lines. Briefly, 50,000 cells were plated in 6-cm plates, trypsinized and viable cell counts were determined using the ViCell automated cell counting system. Each time point was performed in duplicate, and lysates were prepared from the day 7 time point to confirm high TXNIP expression using Western blot analysis. Each experiment was performed at least three times. Mean and SEM are plotted. Closed squares () indicate vector control cells, and open diamonds (*) indicate cells with TXNIP overexpression. In vitro invasion assays were performed on the TXNIP-overexpressing stable HTh74 (C) and T238 (D) cell lines as described in the Methods section. Results from three independent experiments were combined and normalized to the vector control average and graphed with mean plus SEM. *p

Supportive validation

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Experimental details: Figure 5 Overexpression of TXNIP activates NLRP3-inflammasome and EC inflammation in an IL-1beta-dependent fashion. Representative WB blots ( a , e ) and statistical analysis of protein expression detected from REC lysates, of endothelial ( b ) NLRP3, ( c ) cleaved caspase-1, ( d ) cleaved IL-1beta, and ( f ) TNF-alpha, showing significant increases in NLRP3, cleaved IL-1beta and TNF-alpha and a trend towards increased expression of cleaved caspase-1 in the TXNIP overexpressing (TXNIP ++ + vehicle) group compared with the EV-GFP + vehicle control group. Treatment of TXNIP-overexpressing REC with 100 ng/mL of IL-1RA (TXNIP ++ + IL-1RA) suppressed the effect of TXNIP overexpression on inflammasome activation and TNF-alpha expression. Two-way ANOVA showed significant interaction between IL-1RA and TXNIP overexpression across NLRP3 and cleaved IL-1beta expression levels ( n = 5-6, each measurement was performed in triplicate using two different cell preparations; * p < 0.05 vs. EV-GFP + vehicle group).

Supportive validation

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Experimental details: Figure 6 Silencing of TXNIP abolishes, while its overexpression induces, REC adhesion molecule upregulation in an IL-1beta-dependent fashion. ( a ) Representative WB blots (boxes denote separate lanes from the same gel) and statistical analyses of ( b ) ICAM-1 and ( c ) PECAM-1 protein expression showed higher levels in cells incubated with PAL-BSA compared with BSA controls in the scrambled-siRNA group. Silencing TXNIP expression using siRNA abolished such responses. Two-way ANOVA showed significant interaction between Pal-BSA and silencing TXNIP expression across PECAM-1 levels ( n = 3-4, each measurement was performed in duplicate using two different cell preparations; * p < 0.05 vs. SCsiRNA + BSA, # vs. SCsiRNA + Pal-BSA). ( d ) Representative blots and WB analysis of ( e ) ICAM-1 and ( f ) PECAM-1 protein expression showed higher levels in TXNIP overexpression (TXNIP ++ + vehicle) group compared with EV-GFP + vehicle control group, respectively. Treatment of TXNIP-overexpressing REC with 100 ng/mL of IL-1RA (TXNIP ++ + IL-1RA) suppressed the effect of TXNIP overexpression on expression of REC adhesion molecules ( n = 5-6, each measurement was performed in triplicate using two different cell preparations; * p < 0.05 vs. EV-GFP+ vehicle, # vs. TXNIP + + + vehicle).

Supportive validation

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Experimental details: Figure 2 TXNIP upregulation is responsible for ROS accumulation in HUVEC cultured in high glucose levels. ( A ) HUVEC were cultured in medium containing 5 mM (CTR), 11.1 mM or 30 mM of d -glucose after TXNIP silencing. A scrambled non-silencing sequence (NS) was used as a control for silencing. Western blot (upper panel) was performed on cell lysates using specific antibodies against TXNIP. Actin was used an equal loading control. A representative blot is shown. Densitometric analysis (lower panel) was performed using Image J Lab software on three different blots, and the results are the mean of three independent experiments +- SD. ( B ) HUVEC were cultured in medium containing 5 mM (CTR), 11.1 mM or 30 mM of d -glucose (-) and either after TXNIP silencing or the addition of NAC 5 mM. l -glucose (30 mM) was used as a control of osmolarity. ROS production was evaluated by DCFH, as described in the methods. The results are the mean of three experiments performed in triplicate +- SD. * p < 0.05; ** p < 0.01; *** p < 0.001.

Supportive validation

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Experimental details: Figure 3 VitD prevents TXNIP upregulation and ROS accumulation in HUVEC cultured in high glucose conditions. ( A ) Western blot (upper panel) was performed on cell lysates using specific antibodies against TXNIP. Actin was used as an equal loading control. A representative blot is shown. Densitometric analysis (lower panel) was performed using Image J Lab software on three different blots, and the results are the mean of three independent experiments +- SD. ( B ) HUVEC were cultured in medium containing 5 mM (CTR), 11.1 mM, or 30 mM of d -glucose in the absence (-) or in the presence of VitD 20 nM. l -glucose (30 mM) was used as a control of osmolarity. ROS production was evaluated by DCFH, as described in the methods. The results are the mean of three experiments performed triplicate +- SD. * p < 0.05; ** p < 0.01; *** p < 0.001.

Supportive validation

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Experimental details: Figure 2. TXNIP translocates to the plasma membrane following H 2 O 2 stimulation, assayed by plasma membrane sheet. A and B, TXNIP present in plasma membrane sheets was detected by immunoepifluorescence microscopy for the indicated time. Con indicates control. C, Western blot analysis of cell fractionation indicated that the majority of TXNIP under basal conditions is nuclear and translocates to the membrane with H 2 O 2 treatment. Western blotting of organelle-specific markers demonstrated the purity of cell fractions.

Supportive validation

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Experimental details: Figure 6. TXNIP promotes cell survival that requires TXNIP-TRX1 binding. A, Apoptosis assayed by nuclear morphology of EC transfected with TXNIP siRNA, rescued with either TXNIP-WT-GFP or TXNIP-Cys247Ser-GFP, and treated with H 2 O 2 for 6 hours. &ast P

Supportive validation

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Experimental details: Figure 7 PTEN and TXNIP proteins level changes after exogenous miR-20b expression inhibition. PTEN ( A ) and TXNIP ( B ) protein expression comparison 48 h and 72 h after transfection in AGS and MKN28 cell cultures transfected with anti-miR-20b and miR-control. Significant PTEN protein level increase was determined 72 h after transfection in AGS cell culture and TXNIP- 72 h after transfection in MKN28 cell culture. Representative pictures of PTEN and TXNIP proteins signals detected by Western Blot presented at the bottom of a figure (* p < 0.05). Data from four independent experiments.

Supportive validation

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Experimental details: 2 Fig. Doxycycline-induced TXNIP suppresses proliferation of MLL-r AML cells. (A) Expression of TXNIP in doxycycline (dox)-on-dependent TXNIP-overexpressed MOLM-13 and MV4-11 cells. Cells were treated with or without 3 mu m doxycycline. Seventy-two hours after treatment, cell lysates were prepared and analyzed by immunoblotting with the indicated antibodies. GAPDH was used as loading control. Images are representative images of three reproducible independent results. (B) Growth curves of dox-on-dependent TXNIP-overexpressed MOLM-13 and MV4-11 cells treated with or without doxycycline ( n = 3). Data are expressed as the mean +- SEM values. ** P < 0.01, *** P < 0.001, by 2-tailed Student's t- test.

Supportive validation

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Experimental details: Figure 4 Dysregulation of realtime polymerase chain reaction. It shows significant (A) reduction in miR-17-5p and (B) increase in TXNIP mRNA levels in mice kept on HFD for 4 wk compared to ND, which were reversed with PBA treatment ( n = 3-4); C: Representative western blot were cut from the same membrane for TXNIP and alphatubulin from retina (D) Statistical analysis showed an upregulation in TXNIP expression in HFD mice compared to ND, PBA treatment ified this effect ( n = 4-5) ( a P < 0.05 vs ND, c P < 0.05 vs HFD + PBA) (E) Representative western blot of TXNIP and GAPDH from rMC1 treated with palmitate (pal), after the addition of IRE inhibitor or PBA; D: Statistical analysis showed a trend increase in TXNIP expression, which is reversed by IRE inhibitor or PBA ( P = 0.076, n = 3). HFD: High fat diet; ND: Normal diet; PBA: Phenyl-butyric acid; TXNIP: Thioredoxin-interacting protein; CT: Control; PAL: Palmitate; PAL/IRE1: Palmitate + inositol requiring enzyme 1; PAL/PBA: Palmitate + phenyl-butyric acid.