MA5-13426

antibody from Invitrogen Antibodies

Targeting: AR AIS, DHTR, HUMARA, NR3C4, SBMA, SMAX1

Provider product page for MA5-13426

Western blot

Antibody data

Antibody Data
Antigen structure
References [71]
Comments [0]
Validations
Western blot [3]
Immunocytochemistry [1]
Immunoprecipitation [1]
Immunohistochemistry [1]
Other assay [12]

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Product number: MA5-13426 - Provider product page
Provider: Invitrogen Antibodies
Product name: Androgen Receptor Monoclonal Antibody (AR 441)
Antibody type: Monoclonal
Antigen: Synthetic peptide
Description: MA5-13426 targets Androgen Receptor in ICC/IF, IHC (P), IP, and WB applications and shows reactivity with human samples.
Reactivity: Human
Host: Mouse
Isotype: IgG
Antibody clone number: AR 441
Vial size: 500 µL
Concentration: 0.2 mg/mL
Storage: 4° C

AR protein structure - MA5-13426 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of Androgen Receptor was performed by loading 50 µg of LNCaP, MCF7, HeLa, and DU145 whole cell lysates, and 10 µL of PageRuler Prestained Protein Ladder (Product # 26616) onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with StartingBlock T20 (Product # 37543) for 1 hour. The membrane was probed with an Androgen Receptor monoclonal antibody (Product # MA5-13426) at a dilution of 1:200 overnight at 4C on a rocking platform, washed in TBS-0.1%Tween-20, and probed with an HRP-conjugated goat anti-mouse IgG secondary antibody (Product # 31430) at a dilution of 1:20,000 for 1 hour. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34075). Images were acquired on a Thermo Scientific myECL Imager (Product # 62236).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of Androgen Receptor was performed by loading 20 µg of LNCaP nuclear extract (NE), cytoplasmic fraction (Cytopl.) and 10 µL of PageRuler Prestained Protein Ladder (Product # 26616) onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with StartingBlock T20 (Product # 37543) for 1 hour. The membrane was probed with an Androgen Receptor monoclonal antibody (Product # MA5-13426) at a dilution of 1:200 overnight at 4C on a rocking platform, washed in TBS-0.1%Tween-20, and probed with an HRP-conjugated goat anti-mouse IgG secondary antibody (Product # 31430) at a dilution of 1:20,000 for 1 hour. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34075). Images were acquired on a Thermo Scientific myECL Imager (Product # 62236). Note: Nuclear and cytoplasmic fractions were obtained using the Thermo Scientific NE-PER Nuclear and Cytoplasmic Extraction Kit (Product # 78833).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot of Androgen Receptor using Androgen Receptor Monoclonal Antibody (Product # MA5-13426) on LNCaP Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunoprecipitation of Androgen Receptor was performed on LNCaP cells. Antigen-antibody complexes were formed by incubating 250 µg of LNCaP whole cell lysate with 2 µg of an Androgen Receptor monoclonal antibody (Product # MA5-13426) overnight on a rocking platform at 4C. The immune complexes were captured on 50 µL Protein A/G Agarose (Product # 20421), washed extensively, and eluted with 5X Lane Marker Reducing Sample Buffer (Product # 39000). Eluted sample and 20 µg of LNCaP whole cell lysate (loading control) were resolved on a 4-20% Tris-HCl polyacrylamide gel, transferred to a PVDF membrane, and blocked with StartingBlock T20 (Product # 37543) for 1 hour. The membrane was probed with an Androgen Receptor monoclonal antibody (Product # MA5-13426) at a dilution of 1:200 overnight rotating at 4øC, washed in TBST, and probed with an HRP- conjugated goat anti-mouse IgG at a dilution of 1:20,000 for 1 hour. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34075). Images were acquired on a Thermo Scientific myECL Imager (Product # 62236).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunoprecipitation of Androgen Receptor using Androgen Receptor Monoclonal Antibody (Product # MA5-13426) on denatured Human LNCaP Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 3 AR interacts with and is a substrate for the SUMO E3 ligase Hsp27. a Ectopic induction of a hyperSUMO environment in MCF-7 cells with HA-SUMO3 increases the interaction between AR and Hsp27. b AR-Hsp27 interaction complex is enhanced endogenously in the naturally occurring hyperSUMO environment of TamR-7 cells. a - b Interactions with chromatin-bound (CB) AR were analyzed by western blot. c Immunoprecipitation of Hsp27 was performed to assess its binding with AR, Ubc9 and SUMO-2/3 in a cell-free system. d SUMOylation of AR is enhanced under in vitro conditions when recombinant Hsp27 is added. Details are found in (Additional file 1 )

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 PRPF6 promotes growth of prostate cancer cells. A. PRPF6 and AR expressions in prostate cancer-derived cell lines were analyzed by western blot. Protein extractions of different cell lines were subjected to western blot analysis using antibodies against PRPF6 (Proteintech) and AR (Proteintech). GAPDH was used as internal control. B. Effects of PRPF6 knockdown on the colony formation of CWR22Rv1 cells were shown. CWR22Rv1 cells (1000 per well) were infected with lentivirus shRNA targeting PRPF6 (shPRPF6) or control lentivirus (shCtrl), and then were treated with 10 -8 M DHT or ethanol for 14 days. C and D. PRPF6 knockdown repressed cell proliferation. CWR22Rv1 cells (C) and LNCaP (D) (2500 per well) with shPRPF6 or shCtrl were treated with 10 -8 M DHT or ethanol. After indicated time, MTS reagent was added, then absorbance at 490 nm was measured and plotted. Data were means +- SD of three independent experiments. Student's t -test was performed. E. Growth inhibition of prostate cancer cells by PRPF6 was associated with AR. CWR22Rv1 cells (2500 per well) with shPRPF6 or shCtrl were transfected with AR expression lentivirus or control lentivirus, and then were treated with 10 -8 M DHT (right) or ethanol (left). After indicated time, MTS reagent was added, then absorbance at 490 nm was measured and plotted. Data were means +- SD of three independent experiments. Student's t -test was performed. F. Representative photograph of mice with tumor xenografts (upper panel). Pho

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 PRPF6 interacts with AR in prostate cancer cells. A. Exogenous PRPF6 interacted with AR-FL in HEK293 cells. HEK293 cells were co-transfected with plasmids expressing AR-FL and PRPF6 or vector as indicated. After 4 hrs, cells were treated by DHT (10 -8 M) or ethanol vehicle for another 24 hrs. Then cells were harvested and equal amounts of cell lysates were subjected to immunoprecipitation with anti-AR antibody (Invitrogen). The immunoprecipitated proteins were subjected to western blot analysis using anti-AR (Proteintech) and the indicated antibodies. GAPDH was used as internal control. B and C. Endogenous PRPF6 and AR interacted with each other in CWR22Rv1 cells. Upon treatment of 10 -8 M DHT or ethanol vehicle for 24 hrs, CWR22Rv1 cells were harvested and equal amounts of cell lysates were subjected to immunoprecipitation with normal IgG, anti-AR antibody (Invitrogen) or anti-PRPF6 antibody (Santa Cruz). The immunoprecipitated proteins were subjected to western blot analysis using antibodies against PRPF6 (Proteintech) and AR (Proteintech). GAPDH or beta-Actin was used as internal control. D. Diagram of plasmids encoding FLAG-tagged PRPF6-FL and truncated mutants. E. Subcellular localizations of the AR and PRPF6-FL or truncated mutants. HEK293 cells were co-transfected with plasmids expressing AR-FL and PRPF6-FL or truncated mutants. After 10 -8 M DHT stimulation or ethanol vehicle for 4 hrs, cells were fixed and stained with antibody against FLAG and AR (Proteinte

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 Mechanisms involved in promotion of AR-mediated transactivation by PRPF6. A, B and C. PRPF6 knockdown impacted on the recruitments of PRPF6 and AR, as well as the epigenetic modification on AREI/II (A), AREIII (B) in PSA and ARE (C) in KLK2 . CWR22Rv1 cells infected with shPRPF6 or shCtrl were treated with 10 -8 M DHT or ethanol for 4 hrs, then cells were subjected to ChIP assays with using normal IgG, anti-AR (Invitrogen), anti-PRPF6 (Bethyl) or the indicated antibodies. D. PRPF6 interacts with JMJD1A in CWR22Rv1 cells. Upon treatment of 10 -8 M DHT or ethanol vehicle for 24 hrs, CWR22Rv1 cells were harvested and equal amounts of cell lysates were subjected to immunoprecipitation with normal IgG, anti-PRPF6 antibody (Proteintech). The immunoprecipitated proteins were subjected to western blot analysis using antibodies against PRPF6 (Proteintech), MLL1 (Bethyl) and JMJD1A (Proteintech). beta-Actin was used as internal control. E and F. PRPF6 knockdown reduced the interaction between JMJD1A and AR. CWR22Rv1 cells infected with shPRPF6 or shCtrl were transfected with AR expression plasmid (in F) or not (in E). Then, upon treatment of 10 -8 M DHT or ethanol vehicle for 24 hrs, cells were harvested and equal amounts of cell lysates were subjected to immunoprecipitation with normal IgG, anti-AR antibody (Invitrogen). The immunoprecipitated proteins were subjected to western blot analysis using antibodies against PRPF6 (Proteintech), JMJD1A (Proteintech) and AR (Proteintec

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 PRPF6 promotes the expressions of AR-FL and AR-V7. A and B. Knockdown of PRPF6 downregulated mRNA expressions of AR-FL in LNCaP cells (A), and downregulated mRNA expression of AR-FL and AR-V7 in CWR22Rv1 cells (B). Cells were transfected with two independent siRNAs against PRPF6 (siPRPF6) or control siRNA (siCtrl). After 48 hrs, cells were collected for RNA extraction and real-time PCR were performed. Student's t -test was performed. C. Knockdown of PRPF6 downregulated protein expressions of AR-FL and AR-Vs in CWR22Rv1 cells. CWR22Rv1 cells were transfected with siCtrl or siPRPF6. After 48 hrs, cells were collected for protein extraction and subjected to western blot analysis using antibodies against PRPF6 (Santa Cruz) and AR (Proteintech). beta-Actin was used as internal control. D. Ectopic expression of PRPF6 upregulated protein expressions of AR-FL and AR-Vs in CWR22Rv1 cells. CWR22Rv1 cells were transfected with different amounts of Myc-tagged PRPF6 expression plasmids or control vector. After 48 hrs, cells were collected for protein extraction and subjected to western blot analysis using anti-AR (Proteintech) and the indicated antibodies. GAPDH was used as internal control. E and F. Impacts of lentivirus-mediated PRPF6 knockdown on the expression of AR-FL in LNCaP cells. LNCaP cells with shPRPF6 or shCtrl were collected for RNA extraction and real-time PCR were performed (E), or protein extraction and subjected to western blot analysis using antibodies against P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 6 PRPF6 promotes cell growth under androgen-depleted condition. A. Impacts of lentivirus-mediated PRPF6 knockdown on the protein expressions in LNCaP-AI cells. LNCaP-AI cells were infected with shPRPF6 or shCtrl, and then were collected for protein extraction and subjected to western blot analysis using antibodies using anti-PRPF6 (Proteintech), anti-AR (Proteintech) and GAPDH. B. PRPF6 knockdown repressed cell proliferation of LNCaP-AI cells. LNCaP-AI cells (2500 per well) with shPRPF6 or shCtrl were cultured in medium with 5% CSS. After indicated time, MTS reagent was added, then absorbance at 490 nm was measured and plotted. Data were means +- SD of three independent experiments. Student's t -test was performed. C. PRPF6 knockdown inhibited cell growth of LNCaP-AI cells under androgen-depleted condition. LNCaP-AI cells (5000 pre well) with shPRPF6 or shCtrl were infected with shPRPF6 or shCtrl, and then were cultured for 14 days. D. PRPF6 knockdown inhibited cell growth in xenograft mice under castration condition. Castration operation was performed on 5-week-old male NOD/SCID mice. After one week, CWR22Rv1 cells with shPRPF6 or shCtrl were injected subcutaneously into castrated male mice. Representative photograph of mice with tumor xenografts (upper panel). Photograph of all xenograft tumors derived from CWR22Rv1 cells with shPRPF6 or shCtrl (lower panel). E. Tumor volumes of xenografts in castrated male mice were shown. The volume of xenograft tumors derived from

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3--figure supplement 3. AR and ERG expression in RWPE-1, and mutation of AR LBD. ( B ) RWPE-1 control cells and stably expressing AR wild type (AR WT) were either left untreated ( C ) or treated with 100ng/ml doxycycline ( D ) to induce ERG expression, in the absence ( D ) or presence (R/D) of 1nM R1881 for 24 hr. Western blot analysis shows expression levels of AR and ERG. ( B ) qRT-PCR of the luminal genes PSA (blue) and NKX3-1 (red) from RWPE-1 cells treated as in ( A ). Data represent normalized expression of PSA and NKX3-1 mRNA relative to the B2M transcript. Error bars represent + SEM of three replicates. ( C ) Western blot analysis of AR, ERG and GAPDH expression levels from RWPE-1 parental cells or cells stably expressing either AR wild type (WT), R711K, R727K, R753K, R761K, R775K, R780K, R787K, R789K, R787/789K, R832K, R841K, R847K, R855K, R856K, R855/856K or R872K AR mutant. ( D ) RWPE-1 cells stably expressing either AR R711K, R727K, R753K, R775K, R780K, R787K, R789K, R787/789K, R832K, R841K, R847K, R855K, R856K, R855/856K or R872K mutant were used for AR IP followed by western blot analysis for MMA, SDMA and total AR levels. DOI: http://dx.doi.org/

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3. PRMT5 methylates AR on arginine 761. (A) ( A ) Left panel: western blots of noted proteins from VCaP whole cell extracts (WCE) after ERG or PRMT5 knockdown. Right panel: Western blot of AR immunoprecipitation (IP) from VCaP. SDMA: symmetric di-methyl arginine; MMA: mono-methyl arginine. ( B ) Left panel: Western blot analysis of noted proteins from homozygous TMPRSS2:ERG transgenic (Tg/Tg) and WT mouse tissues. Right panel: AR or IgG IP from mouse tissues followed by western blot analysis of MMA, SDMA and total AR levels. ( C ) Western blot of AR immunoprecipitation (IP) from VCaP and LNCaP cells grown in charcoal-stripped serum (CSS) and stimulated with 10nM R1881. SDMA: symmetric di-methyl arginine; MMA: mono-methyla arginine. ( D ) Left panel: western blot of noted proteins from 22Rv1 parental (PAR) or ERG-expressing (ERG) WCEs. FL: full-length; TR: truncated (lacking ligand binding domain, LBD). Right panel: Western blot of AR IP from 22Rv1. ( E ) Left panel: RWPE-1 parental (PAR) and AR and ERG-expressing (AR/ERG) cells targeted by PRMT5 knockdown (PRMT5 shRNA) or NTC shRNA were left either untreated ( C ) or treated with 100ng/ml doxycycline ( D ) in the absence or presence of 1nM R1881 (R) for 24 hr. Western blot analysis shows expression levels of PRMT5, ERG, AR and GAPDH from input samples (WCE). Right panel: Lysates were then used for AR immunoprecipitation (AR IP) followed by western blot analysis using antibodies against MMA, SDMA or total AR levels. ( F

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 2 MiR-346, -361-3p and -197-3p alter wild-type and variant androgen receptor activity in prostate cancer (PC) cells partially through association with AR 3'UTR. a qRT-PCR analysis of AR mRNA levels in C42/MAR4 cells transfected with (i) miR-346, (ii) miR-361-3p or (iii) miR-197-3p mimic and/or inhibitor for 24 h. b Western blot analysis of AR protein levels in C42/MAR4 transfected with (i) miR-346, (ii) miR-361-3p or (iii) miR-197-3p mimic and/or inhibitor for 24 h. beta-actin was used as a control for loading. Representative blots of three independent experiments are shown. Additional biological replicates and densitometry for three independent experiments are shown (Fig. S 1c-g ). c qRT-PCR analysis of PSA mRNA levels in LNCaP/MAR4 cells transfected with (i) miR-346, (ii) miR-361-3p or (iii) miR-197-3p mimic and/or inhibitor for 24 h. L19 was used as a normalisation gene. d , e Luciferase assay analysis of 6.9 kb AR 3'UTR activity in HEK293T ( d ) or C42 ( e ) cells transfected with pMiR-Report vector containing WT ( d ) or miR binding site-mutant ( e ) regions of the AR 6.9 kb 3'UTR as depicted (Fig. S 2Di ) +- miR mimics or inhibitors (20 nM) for 48 h ( d ) or 72 h ( e ) Luciferase activity was normalised to beta-galactosidase activity to correct for transfection efficiency. Columns: mean normalised AR reporter luciferase activity from three independent experiments performed in duplicate +- SEM. f AGO2/biotin-miR RNA-IP analysis of miR-197 and miR-346 association wit

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 7 AR-modulatory miR target siRNA silencing phenocopies miRs. a Western blot analysis of ARHGDIA, TAGLN2, YWHAZ and AR protein levels in LNCaP cells transfected with siRNAs (1 or 10 nM) targeting (i) YWHAZ, (ii) TAGLN2 or (iii) ARHGDIA for 72 h. A minimum of two different siRNAs were used for each gene. beta-actin was used as a control for loading. b qRT-PCR analysis of (i) AR or (ii) PSA mRNA levels in LNCaP cells transfected with ARHGDIA, TAGLN2 or YWHAZ siRNAs (10 nM) for 48 h. L19 was used as a normalisation gene. Columns: mean +- SEM for three independent experiments performed in triplicate. c SRB proliferation assay analysis of LNCaP cells transfected with siRNA targeting ARHGDIA, TAGLN2 or YWHAZ (10 nM) for 6 days. Data are presented relative to absorbance at day 0. Points: mean absorbance at 492 nm for three independent experiments performed in quadruplicate +- SEM. d , e MiR-197 ( d i), miR-346 ( d ii), miR-361-3p levels ( d iii), TAGLN2 ( e i), ARHGDIA ( e ii) and YWHAZ ( e iii) RNA levels in fresh-frozen tissue from five PCs and six normal prostates (GSE34932). f YWHAZ mRNA levels in PC tumours of Gleason Grades 6-10 (i) and miR-346 levels in PC tumours of lymph node-positive and -negative patients from the TCGA-PRAD data set of 333 primary prostate cancers (PCs). g Linear regression analysis of correlation between AR activity and ARHGDIA (i), TAGLN2 (ii) and YWHAZ (iii) levels in CRPC patient tumours ( n = 122, transcriptome data from SU2C-PCF). AR activity sc