PA1-027A

antibody from Invitrogen Antibodies

Targeting: PPIB B, CYP-S1, CYPB, OI9, PPIase, SCYLP

Provider product page for PA1-027A

Western blot

Antibody data

Antibody Data
Antigen structure
References [52]
Comments [0]
Validations
Western blot [4]
Immunocytochemistry [2]
Immunohistochemistry [3]
Flow cytometry [2]
Other assay [24]

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Product number: PA1-027A - Provider product page
Provider: Invitrogen Antibodies
Product name: Cyclophilin B Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: PA1-027A detects cyclophilin B (CyPB) in mouse, rat, canine, and human tissues.
Concentration: 1 mg/mL

PPIB protein structure - PA1-027A shown in red.

Supportive validation

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Experimental details: Western blot analysis of Cyclophilin B in Rat Liver lysate (25 µg) using anti-Cyclophilin B antibody, PA1-027A at a dilution of 1:2000 (lane 2), 1:1000 (lane 3), 1:2000 (lane 4) and 1:4000 (lane 5).

Supportive validation

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Experimental details: Western blot analysis of Cyclophilin B was performed by loading 30 µg of HeLa (Lane 1), HeLa - Cyclophilin B knockout (Lane 2) whole cell lysate. The blot was probed with Anti-Cyclophilin B Antibody (Product # PA1-027A, 1 µg/mL) Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). Loss of signal upon CRISPR mediated knockout (KO) confirms that antibody is specific to Cyclophilin B.

Supportive validation

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Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of U-87 MG (Lane 1), IMR-32 (Lane 2), PC-3 (Lane 3), U-2 OS (Lane 4), SH-SY5Y (Lane 5), Jurkat (Lane 6), and MCF 7 (Lane 7). The blots were probed with Anti-Cyclophilin B Rabbit Polyclonal Antibody (Product # PA1-027A, 1-2 µg/mL) and detected by chemiluminescence using Goat Anti-Rabbit IgG Secondary Antibody, HRP conjugate (Product # G-21234, 1:5000 dilution). A ~ 23 kDa band corresponding to Cyclophilin B was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10 % Bis-Tris gel (Product # NP0302BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

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Experimental details: Knockdown of Cyclophilin B was achieved by transfecting U-87 MG cells with Cyclophilin B specific siRNAs (Silencer® select Product # s10907). Western blot analysis (Fig a) was performed using membrane enriched extracts from the Cyclophilin B knock down cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Anti- Cyclophilin B Rabbit polyclonal Antibody (Product # PA1-027A, 1µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 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 Cyclophilin B.

Supportive validation

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Experimental details: Immunohistochemistry analysis of Cyclophilin B showing staining in the cytoplasm of paraffin-embedded human hepatocarcinoma (right) compared with a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Cyclophilin B polyclonal antibody (Product # PA1-027A) diluted in 3% BSA-PBS at a dilution of 1:200 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of Cyclophilin B showing staining in the cytoplasm of paraffin-embedded human liver tissue (right) compared with a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Cyclophilin B polyclonal antibody (Product # PA1-027A) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of Cyclophilin B showing staining in the cytoplasm of paraffin-embedded rat liver tissue (right) compared with a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Cyclophilin B polyclonal antibody (Product # PA1-027A) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

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Experimental details: Figure 2 miR-100 inhibits tumorigenesis and is downregulated in human breast cancer. (A) miR-100 expression levels in four subtypes of human breast tumors and paired normal breast tissues, based on the RNA-Seq data from TCGA. Statistical significance was determined by paired t test. (B) Representative images of in situ hybridization of miR-100 in normal human mammary glands and human breast carcinomas. Blue color indicates positive staining. (C) miR-100 scores (normalized to U6) based on in situ hybridization of human breast tissue microarray (TMA). (D) Immunoblotting of Erbb2 and cyclophilin B (CypB) in HMLE cells transduced with miR-100 and Erbb2, alone or in combination. (E) Tumor growth by 1.5x10 6 subcutaneously injected HMLE cells transduced with Erbb2 alone or in combination with miR-100. Data are mean +- SEM ( n = 10 mice per group). (F, G) Tumor weight (F) and tumor images (G) of mice with subcutaneous injection of HMLE cells transduced with Erbb2 alone or in combination with miR-100, at day 31 after implantation. Data in (F) are mean +- SEM ( n = 10 mice per group). (H) Immunoblotting of E-cadherin, vimentin and cyclophilin B (CypB) in tumor lysates from (G). (I, J) Tumor weight (I) and tumor images (J) of mice with subcutaneous injection of 5x10 6 miR-100-transduced MCF7 cells, at day 22 after implantation. Data in (I) are mean +- SEM ( n = 7-8 mice per group). Statistical significance in (C), (E), (F) and (I) was determined by two-tailed, unpaired Student's t

Supportive validation

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Experimental details: Figure 3 miR-100 downregulates E-cadherin by targeting SMARCA5 . (A) Immunoblotting of SMARCA5, HOXA1 and HSP90 in HMLE and MCF7 cells transduced with miR-100. (B) Luciferase activity of the wild-type or mutant HOXA1 3' UTR reporter gene in 293T cells with ectopic expression of miR-100. (C) Immunoblotting of SMARCA5, E-cadherin and cyclophilin B (CypB) in HMLE cells infected with the SMARCA5 shRNA (shSMARCA5) or the pLKO.1-puro lentiviral vector with a scrambled sequence (Scr) that does not target any mRNA. (D) qPCR of CDH1 in HMLE cells transduced with the control vector (mock), miR-100 alone or in combination with SMARCA5. (E) Immunoblotting of SMARCA5, E-cadherin and HSP90 in HMLE cells transduced with the control vector (mock), miR-100 alone or in combination with SMARCA5. (F) Bisulfite sequencing assay (left panel) and the percentage of CpG methylation (right panel) of the CDH1 promoter in HMLE cells transduced with the control vector (mock), miR-100 alone or in combination with SMARCA5. Open circles: unmethylated CpG sites; solid black circles: methylated CpG sites. Data in (B), (D) and (F) are mean +- SEM, and statistical significance was determined by two-tailed, unpaired Student's t test.

Supportive validation

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Experimental details: Fig. 1 Comparison of MATR3 protein levels in a panel of basal-like/triple-negative human breast cancer cell lines. a Endogenous MATR3 protein levels were examined in a small panel of basal-like/triple-negative breast cancer cell lines by western blotting. The lower panel demonstrates the relative normalized MATR3 protein levels. b MATR3 was transiently overexpressed in MDA-MB-231 cells and the expression was determined by western blotting. The lower panel demonstrates the relative normalized MATR3 protein levels. c MATR3 was silenced by shRNAs and the expression was determined by western blotting. The lower panel demonstrates the relative normalized MATR3 protein levels. d In vitro cell proliferation was compared between control and MATR3-overexpressing MDA-MB-231 and between control and MATR3-depleted BT549 cells. n = 4 per group. e Anchorage-independent growth was examined to compare in vitro tumorigenesis by soft agar colony formation assay. n = 4 per group. Statistical significance in d and e was determined by an unpaired t -test. Error bars are s.e.m

Supportive validation

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Experimental details: Figure 5. MALAT1 inactivates TEAD ( a ) Luciferase activity in HEK293FT cells co-transfected with indicated plasmids. n = 4 cell culture replicates per group. ( b ) Luciferase activity in control and MALAT1 knockout MDA-MB-231 cells co-transfected with indicated plasmids. n = 3 cell culture replicates per group. ( c, d ) HEK293FT cells were co-transfected with Malat1 , SFB-YAP, and HA-TEAD1, and were subjected to pulldown with S-protein beads ( c ) or an HA-specific antibody ( d ), followed by immunoblotting with antibodies against pan-TEAD and FLAG. ( e ) ChIP-qPCR analysis showing the occupancy of ANKRD1 , CTGF , and CYR61 promoters by TEAD1 or YAP immunoprecipitated from control or MALAT1 knockout MDA-MB-231 cells. ( f ) qPCR of YAP-TEAD target genes in the tumors of MMTV-PyMT; Malat1 +/+ (PyMT;WT), MMTV-PyMT; Malat1 -/- (PyMT;KO), and MMTV-PyMT; Malat1 -/- ; Malat1 Tg (PyMT;KO;Tg) mice. n = 5 mice per group. ( g ) Immunoblotting of pan-TEAD and cyclophilin B (CypB) in MALAT1 knockout MDA-MB-231 cells with or without transduction of TEAD shRNA. Scr: scramble control. ( h, i ) Bioluminescent imaging ( h ) and photon flux quantification ( i ) of NSG mice with intravenous injection of control and MALAT1 knockout MDA-MB-231 cells with or without transduction of TEAD shRNA. Day 0: the day of tumor cell injection. n = 5 mice per group. ( j ) Bioluminescent imaging (upper panel) and photon flux quantification (lower panel) of the lungs from mice described in Fig. 5h, i . n = 5

Supportive validation

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Experimental details: Fig. 2 Important chaperone features for functional replacement of tapasin localize to scaffolding sites for MHC-I. a TAPBPR and variants of tapasin were transfected in tapasin-KO Expi293F cells, and relative surface HLA-A*02:01 expression as measured by flow cytometry is plotted. Data are mean +- SD, n = 4 independent experiments. Proteins were FLAG-tagged at their luminal N-termini and immunoblots of the whole lysate are shown, with cyclophilin B (CycB) used as a loading control. b Tapasin was deep mutationally scanned based on selection of tapasin-KO cells with rescued surface HLA-A*02:01. Log 2 enrichment ratios for mutations across residues 10-20 are plotted from =+3 (enriched, dark blue). Residue position is on the horizontal axis, and amino acid substitutions are on the vertical axis (*, stop codon). c Conservation scores from the entire deep mutational scan of the tapasin/MHC-I interface are mapped to a homology model of HLA-A*02:01-bound tapasin. Highly conserved residues for mediating the folding and surface trafficking of HLA-A*02:01 are colored orange, while neutral regions are pale white/blue. Residues excluded from the library and analysis are gray. MHC-I H chain and hbeta 2 m are pale and dark green ribbons, respectively. d A chimera of the TAPBPR luminal domain with the tapasin TM and cytosolic domains, called TAPBPR-CT, has increased activity for chaperoning endogenous HLA-A*02:01, despite reduced protein expression by i

Supportive validation

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Experimental details: Figure 2. Rocaglate-inducible GEF-H1 regulates JNK signaling via RHOA (A) Schematic of RHOA/JNK activation by rocaglate-induced GEF-H1 expression. (B) Relative total cell number of U87MG treated with indicated siRNAs (pool of four independent siRNAs for each target) for 48 h, followed by silvestrol treatment at indicated concentrations or vehicle (DMSO) for 24 h. Data represent mean +- SEM (error bars) of three independent experiments. *p < 0.05 compared to NS (non-silencing) siRNA control. (C) Representative immunoblots of U87MG treated with indicated concentrations of silvestrol, rocaglamide A, or vehicle (DMSO) for 24 h. (D) Representative immunoblots of U87MG treated with indicated siRNA pools for 48 h, followed by silvestrol treatment at indicated concentrations or vehicle (DMSO) for 24 h. (E) Representative immunoblots of U87MG treated with indicated 3' UTR-targeting siRNA pools and expression construct simultaneously for 48 h, followed by treatment with silvestrol (6.25 nM) or vehicle (DMSO) for 24 h. GEF-H1 ORF, coding region-only construct; short, short exposure; long, long exposure. (F) Representative immunoblots of U87MG treated with indicated siRNA pools (100 nM final concentration) for 72 h, followed by treatment with silvestrol (12.5 nM) or vehicle (DMSO) for 24 h. Densitometry values in (C) and (D) are mean of three independent experiments, (E) and (F) are normalized to loading control. (G) Representative immunoblots of active RHO (RHO-GTP) pull-down lysates fr

Supportive validation

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Experimental details: Figure 3. Rocaglate-inducible JNK phosphorylation mediates cellular toxicity signaling (A) Representative immunoblots of U87MG treated with silvestrol (12.5 nM), rocaglamide A (rocA, 12.5 nM), or vehicle (DMSO) with and without JNK inhibitor JNK-IN-8 (10 muM) for 24 h. Densitometry values (mean of three independent experiments) are normalized to loading control. (B and C) Relative cell viability based on ATP levels in U87MG treated with indicated concentrations of (B) silvestrol (left panel) and rocaglamide A (right panel) or (C) zotatifin with and without JNK inhibitor JNK-IN-8 (10 muM) for the indicated durations. For (B) and (C), data represent mean +- SEM (error bars) of four independent cell populations. *p < 0.05, **p < 0.01, ***p < 0.001 compared to corresponding rocaglate only condition. (D) Representative immunohistochemistry images (D; scale bars represent 50 mum) and immunoblots (E) of glioblastoma (left panel) and DLBCL (right panel) patient-derived xenograft mouse tumors treated with silvestrol (1 mg/kg) or vehicle (2-hydroxypropyl-beta-cyclodextrin) (daily intraperitoneal injection for 3 days). For (E), densitometry values are normalized to loading control. See also Figure S3 .

Supportive validation

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Experimental details: Figure 4. Mechanisms of rocaglate-dependent protein induction (A) mRNA translation efficiency (abundance ratio of polysome-associated mRNA to ribosome-free and monosome-associated mRNA) in U87MG treated with silvestrol (6.25 nM) for 24 h. Data represent mean +- SEM (error bars) of five independent experiments. *p < 0.05 compared to vehicle (DMSO) treatment. (B) Steady-state mature mRNA (left panel) and pre-mRNA (right panel) levels in U87MG treated with silvestrol (6.25 nM) for 24 h. Data represent mean +- SEM (error bars) of five independent experiments. *p < 0.05 compared to vehicle (DMSO) treatment. Data are normalized against 18S rRNA. (C and D) Steady-state mRNA levels (C) and representative immunoblots (D) of U87MG treated with silvestrol (6.25 nM) for 24 h with and without actinomycin D (Act. D, 1 mug/mL, for the last 6 h of silvestrol treatment). Densitometry values in (D) are normalized to loading control. Data in (C) represent mean +- SEM (error bars) of three independent experiments. *p < 0.05 compared to no Act. D treatment. (E and F) Representative immunoblots of U87MG treated with (E) DMDAPatA (indicated concentrations), (F) hippuristanol (100 nM), or vehicle (DMSO) for 24 h. Densitometry values in (E) and (F) are normalized to loading control. (G) Relative total cell number of U87MG treated with DMDAPatA (6.25 nM) (left panel), hippuristanol (100 nM) (right panel), or vehicle (DMSO) for 24 h. Data represent mean +- SEM (error bars) of three independent experime

Supportive validation

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Experimental details: Figure 5. System-wide survey of rocaglate-dependent changes in the translation machinery (A) MATRIX workflow of silvestrol treatment in U87MG. Polysome fractions reflect productive, high-intensity protein synthesis, whereas ribosome-free fractions are associated with translational inactivity. Representative immunoblots of U87MG input samples for ribosome density fractionation, lysed with polysome lysis buffer, are shown at the bottom right. (B-F) MATRIX analysis of silvestrol-induced changes in translational activity (i.e., ratio of protein abundance in polysome versus ribosome-free [free] fraction) for (B) canonical translation factors, (C) ribosomal proteins, (D) most highly induced proteins (>=3-fold activity enrichment), (E) aminoacyl-tRNA synthetases (AARSs), and (F) DEAD/DEAH RNA helicases. Blue and red indicate silvestrol-repressed and silvestrol-activated translational assets (>=2-fold difference in activity), respectively. See also Figure S5 .

Supportive validation

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Experimental details: Fig. 5 Immunostaining of CypB in mock- and HCoV-229E-infected Huh7 cells in the presence of ETOH solvent or cyclophilin inhibitors CsA and Alisporivir . After infection (MOI = 1) medium was removed and new medium was added to cells containing 20 muM of inhibitor for 48 h and samples were processed for IF. CypB and dsRNA were stained with anti-CypB (green) and anti-dsRNA J2 (red, Scicons, 1:1500), respectively. Nuclei were visualized with DAPI. CypB shifts from an even cytoplasmic distribution to intense bleb-like structures in the presence of virus (white arrows, ETOH solvent panels). CsA and ALV led to the re-localization of CypB to granular structures in the nucleus and to a massive reduction of expression independent of virus infection. dsRNA as replication marker was not detected in the presence of CsA or ALV. Exposure times for the respective antibodies were the same in the different samples. Fig. 5

Supportive validation

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Experimental details: Fig. 6 Co-immunostaining of CypB and cell organelles in HCoV-229E-infected Huh7 cells . For the identification of the intense cyclophilin B bleb-like structures infected cells (MOI = 1; 48 h p.i.) were co-stained with anti-CypB and antibodies directed against markers of the ER (anti-PDI), cis-GOLGI (anti-GM130), autophagosomes (anti-SQSTM1), anti-P-bodies (anti-hDcp1a) and stress granules (anti-PABP). Cyclophilin B normally distributes within the ER. In the presence of HCoV-229E, it intriguingly concentrates at bleb-like structures of the ER. Fig. 6

Supportive validation

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Experimental details: Fig. 7 Downregulation of Cyp B, but not CypA in the presence of Cyp inhibitors CsA and ALV. Huh7 cells were either mock or HCoV-229E (MOI = 1) infected and cultivated in the presence of EtOH solvent, or 20 muM CsA or ALV for 48 h. Cell extracts were subjected to Western blot analysis and staining with anti-Cyp A, anti-Cyp B and anti-beta actin as loading control. Fig. 7

Supportive validation

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Experimental details: Figure 3. Activity-dependent Arc expression is blocked in the absence of CREB phosphorylation at Ser133 as well as at Ser142/143. A , Stimulus and virus infection conditions are as follows (from left): ACSF control media in cells infected with HSV-GFP control virus ( n = 105 cells on 12 coverslips from 3 independent experiments), KCl-rich ACSF to depolarize cells infected with HSV-GFP control virus ( n = 128 cells on 15 coverslips from 3 independent experiments), KCl-rich ACSF in cells infected with CREBdn-S133A ( n = 51 cells on 20 coverslips from 3 independent experiments), and KCl-rich ACSF in cells infected with CREBdn-S142A/S143A ( n = 63 cells on 15 coverslips from 3 independent experiments). Top panels show Arc staining in red and bottom panels show HSV infection in green. B , A cumulative distribution plot showing all analyzed cells in each condition clearly displays the activity-dependent increase in Arc expression that is blocked by the CREBdn viruses [one-way ANOVA, F (3,333) = 53.1, p < 0.0001; Tukey's post hoc test GFP(+KCl) vs all other groups p < 0.0001]. C , Representative case of Arc expression in mice injected with CREBdn-S142A/S143A or control GFP. Cyclophilin B (CyB) was used as a loading control. D , Quantification shows a significant reduction in Arc expression after blocked phosphorylation of CREB at Ser142/143 ( t = 2.32, * p = 0.04; df = 8, t test for unequal variances). All error bars indicate standard error of the mean (SEM). OD = optical density.

Supportive validation

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Experimental details: Loss of both Mfn1 and Mfn2 in beta-cells impairs glucose tolerance and glucose-stimulated insulin release. a Expression of Mfn1 and Mfn2 by Western blot (WB) in islets isolated from 8-10-week-old Ctrl, beta-Mfn1 KO , beta-Mfn2 KO and beta-Mfn1/2 DKO mice. Cyclophilin B serves as a loading control. Representative of six independent mice/group for Mfn1 and 10 independent mice/group for Mfn2. Band intensity of each lane of displayed representative blot (shown as fold change compared to Ctrl, expression normalized to cyclophilin B) above each blot in red. b Densitometry (normalized to cyclophilin B) from studies in Fig. 1A. n = 6/group for Mfn1 and 10/group for Mfn2. Data are presented as Mean +- SEM. * p < 0.0005 by one-way ANOVA followed by Tukey's post-hoc test for multiple comparisons. c Blood glucose concentrations measured during IPGTT of 8-week-old Ctrl ( n = 28), beta-Mfn1 KO ( n = 7), beta-Mfn2 KO ( n = 6) and beta-Mfn1/2 DKO ( n = 15) littermates. Data are presented as Mean +- SEM. ( p < 0.0005 by two-way ANOVA for beta-Mfn1/2 DKO mice vs Ctrl followed by Sidak's post-hoc test for multiple comparisons). d Serum insulin concentrations ( n = 7-24/group) measured during in vivo glucose-stimulated insulin release testing in 8-week-old Ctrl ( n = 24), beta-Mfn1 KO ( n = 7), beta-Mfn2 KO ( n = 10) and beta-Mfn1/2 DKO ( n = 14) littermates. Data are presented as mean +- SEM. * p < 0.0005 for beta-Mfn1/2 DKO mice vs Ctrl and beta-Mfn1 KO , p = 0.004 for beta-Mfn1/2 KO vs beta-M

Supportive validation

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Experimental details: Fig 1 Western blot analysis for lysine modifying enzymes and chaperone complex components in skin obtained from wild type (WT) and CypB KO (KO) mice. The protein levels in WT and KO were assessed by their immunoreactivities with the respective antibodies (Ab) relative to that of GAPDH. (A) CypB, (B) LH1, (C) LH2, (D) LH3, (E) GLT25D1, (F) Fkbp65, (G) Sc65, (H) P3h3, (I) Bip, and (J) Hsp47. *p