Anti-Androgen Receptor/AR Antibody Picoband®

Western blot analysis of Androgen Receptor/AR using anti-Androgen Receptor/AR antibody (A00542).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50ug of sample under reducing conditions.
Lane 1: human K562 whole cell lysates,
Lane 2: human U20S whole cell lysates,
Lane 3: human HEK293 whole cell lysates,
Lane 4: human PC-3 whole cell lysates,
Lane 5: human 22RV1 whole cell lysates,
Lane 6: human HepG2 whole cell lysates,
Lane 7: human CACO-2 whole cell lysates.
After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-Androgen Receptor/AR antigen affinity purified polyclonal antibody (Catalog # A00542) at 0.25 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for Androgen Receptor/AR at approximately 120KD. The expected band size for Androgen Receptor/AR is at 120KD.

Immuno-expression of AR (A:3.5yr.) and ERβ (B: 0.1yr., C: 3.5yr. and D: 8yr.) in testes. Scale bar = 50 μm.
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Immunohistochemical analysis of dorsolateral prostate E-cadherin, Vimtein, ERα and AR expression in aged rats. The expression of vimentin, ERα and AR increased, and the expression of E-cadherin decreased in BPA-treated groups. ( a – p ) Representative sections of comparable regions are shown for vehicle control rats ( a , e , i , m ), and animals exposed to BPA (10 μg/kg/day) ( b , f , j , n ), BPA (30 μg /kg/day) ( c , g , k , o ), and BPA (90 μg/kg/day) ( d , h , l , p ) (scale bar: 50 μm, ×400). BPA: bisphenol A; AR: androgen receptor; ERα:estrogen receptor-α.
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IF analysis of Androgen Receptor/AR using anti-Androgen Receptor/AR antibody (A00542).
Androgen Receptor/AR was detected in immunocytochemical section of T47D cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5μg/mL rabbit anti-Androgen Receptor/AR Antibody (A00542) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG (BA1127) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Immunohistochemical analysis of PCNA in DLP. Representative sections of comparable regions were shown for vehicle control rats ( a ), and animals exposed to BPA (10 μg/kg/day) ( b ), BPA (30 μg/kg/day) ( c ), and BPA (90 μg/kg/day), ( d ) (scale bar: 20 μm, ×400).
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Flow Cytometry analysis of A549 cells using anti-Androgen Receptor/AR antibody (A00542).
Overlay histogram showing A549 cells stained with A00542 (Blue line).To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-Androgen Receptor/AR Antibody (A00542, 1μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1μg/1x10⁶) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.

Flow Cytometry analysis of C6 cells using anti-Androgen Receptor/AR antibody (A00542).
Overlay histogram showing C6 cells stained with A00542 (Blue line).To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-Androgen Receptor/AR Antibody (A00542, 1μg/1x106 cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10μg/1x106 cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1μg/1x106) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.

Flow Cytometry analysis of RAW264.7 cells using anti-Androgen Receptor/AR antibody (A00542).
Overlay histogram showing RAW264.7 cells stained with A00542 (Blue line).To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-Androgen Receptor/AR Antibody (A00542, 1μg/1x106 cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10μg/1x106 cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1μg/1x106) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.

Immunoexpression of testosterone (A: 0.1yr, B: 3.5yr and C: 8yr) and estradiol (D: 0.1yr, E: 3.5yr and F: 8yr) in testes from birth to adulthood. Upper insert on panel A: negative control. Black arrows represent spermatozoa. Scale bar = 50 μm.
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Sirtuin 6 plays a key role in AR-induced mitochondrial dysfunction and tubular cell apoptosis in vitro. A – D Representative western blot ( A ) and graphical representations of ( B ) Bax, ( C ) cleaved caspase-3 and ( D ) PGC-1α protein expression levels are shown. * P < 0.05, ** P < 0.01 versus control group ( n = 3); † P < 0.05, ††† P < 0.001 versus STS group ( n = 3). HKC-8 were treated with STS (1 μMol/L) alone or co-treated with DHT (10 μMol/L) for 10 h. E , F TUNEL assay ( E ) and quantitative data ( F ) showed the degree of cellular apoptosis in different groups, as indicated. Frozen kidney sections were stained by TUNEL staining. Arrows indicate positive staining. Scale bar, 75 μm. *** P < 0.001 versus control group ( n = 3); ††† P < 0.001 versus STS group ( n = 3). G – L Representative western blot ( G ) and graphical representations of ( H ) FAS-L, ( I ) Bax, ( J ) cleaved caspase-3, ( K ) Sirtuin 6 and ( L ) PGC-1α protein expression levels are shown. HKC-8 is transfected with control-shRNA or AR-shRNA before H/R injury. * P < 0.05, ** P < 0.01, *** P < 0.001 versus control-shRNA group ( n = 3); † P < 0.05, †† P < 0.01, ††† P < 0.001 versus control-shRNA/H/R group ( n = 3). M – P Representative western blot ( M ) and graphical representations of ( N ) Bax, ( O ) cleaved caspase-3 and ( P ) PGC-1α protein expression levels are shown. HKC-8 cells were transfected with Sirtuin 6 overexpression plasmid and treated with STS (1 μMol/L) or co-treated with DHT (10 μMol/L) for 6 h. * P < 0.05, ** P < 0.01 ( n = 3); †† P < 0.01, ††† P < 0.001 ( n = 3). Q Androgen binds to the AR in cytoplasm, contributing to the translocation of AR from the cytoplasm into cell nucleus. The AR binds to Sirtuin 6 promoter and deregulates Sirtuin 6 expression. The decreased expression of Sirtuin 6 leads to decreased deacetylation of PGC-1α, contributing to decreased transcriptional activity of PGC-1α. It results in downregulation of mitochondrial biogenesis relative genes, such as TFAM, contributing to the imbalance of mitochondrial homeostasis, which further results in renal TECs apoptosis after AKI.
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( a ) Effect on height of dorsolateral prostatic epithelium. After aged rats were treated with 10–90 μg/kg BPA for 3 months, BPA significantly increased the height of DLP epithelium in a dose-dependent way, * P < 0.01, compared with the vehicle controls. BPA: bisphenol A. ( b ) The expression of the PCNA in DLP. The expression of PCNA was increased obviously in BPA-treated groups. * P < 0.05: compared with the vehicle controls; * P < 0.01, compared with the vehicle controls. BPA: bisphenol A.
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Effects of BPA on E 2 , T, and Insulin serum levels in aged male rats. After aged rats were treated with 10–90 μg/kg BPA for 3 months, 90 μg/kg BPA significantly increased the E 2 level and the estrogen to androgen ratio; BPA had the trend of decreasing the T level and increasing the insulin level. p < 0.05, compared with the vehicle controls. BPA: bisphenol A.
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The ectopic knockdown of AR ameliorates renal injury and mitochondrial dysfunction upon IRI. A Experimental design. Green arrow showed the injection of control-shRNA (pLVX-shRNA) or AR-shRNA (pLVX-shAR) plasmid. Male mice were subjected to IRI or sham respectively, and euthanized 24 h after IRI. B Scr levels in three groups, as indicated. Scr was expressed as milligrams per deciliter. ** P < 0.01 versus sham controls ( n = 5); †† P < 0.01 versus control-shRNA group ( n = 5). C BUN levels in three groups, as indicated. BUN was expressed as milligrams per deciliter. ** P < 0.01 versus sham controls ( n = 5); † P < 0.05 versus control-shRNA group ( n = 5). D , E Representative western blot ( D ) and graphical representations of ( E ) AR protein expression levels are shown. *** P < 0.001 versus sham controls ( n = 5); †† P < 0.01 versus control-shRNA group ( n = 5). F , G Representative western blot ( F ) and graphical representations of ( G ) Sirtuin 6 protein expression levels are shown. ** P < 0.01 versus sham controls ( n = 5); †† P < 0.01 versus control-shRNA group ( n = 5). H Representative micrographs showing the expression of Sirtuin 6 in different groups. Paraffin sections were stained with an antibody against Sirtuin 6. Arrows indicate positive staining. Scale bar, 50 μm. I Representative micrographs show renal tubular morphologic injury, the expression of KIM-1 and caspase-3, and TUNEL assay in different groups, as indicated. Paraffin sections were subjected to PAS staining, stained with an antibody against KIM-1 and caspase-3. Frozen kidney sections were subjected to TUNEL staining. Arrows indicate positive staining. Scale bar, 50 μm. (J – N) Representative western blot ( J ) and graphical representations of ( K ) KIM-1, ( L ) FAS-L, ( M ) Bax and ( N ) cleaved caspase-3 protein expression levels are shown. ** P < 0.01, *** P < 0.001 versus sham controls ( n = 5); †† P < 0.01, ††† P < 0.001 versus control-shRNA group ( n = 5). O Tubular injury scores depending on PAS staining in three groups, as indicated. *** P < 0.001 versus sham controls ( n = 5); ††† P < 0.001 versus control-shRNA group ( n = 5). P Representative micrographs showing the expression of PGC-1α and TOMM20 in different groups, as indicated. Frozen kidney sections were stained with an antibody against PGC-1α and TOMM20. Arrows indicate positive staining. Scale bar, 50 μm. Q – S Representative western blot ( Q ) and graphical representations of ( R ) PGC-1α and ( S ) TOMM20 protein expression levels are shown. *** P < 0.001 versus sham controls ( n = 5); ††† P < 0.001 versus control-shRNA group ( n = 5).
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The ectopic expression of Sirtuin 6 relieves renal injury and mitochondrial dysfunction upon IRI. A Experimental design. Green arrow showed the injection of pcDNA plasmid or pFlag-Sirtuin 6 overexpression plasmid. Male mice were subjected to IRI or sham respectively, and euthanized 24 h after IRI. B Scr levels in three groups, as indicated. Scr was expressed as milligrams per deciliter. *** P < 0.001 versus sham controls ( n = 5); †† P < 0.01 versus pcDNA group ( n = 5). C Representative western blot of flag tag is shown. D Representative micrographs showing the expression of PGC-1α and TOMM20 in different groups, as indicated. Frozen kidney sections were stained with an antibody against PGC-1α and TOMM20. Arrows indicate positive staining. Scale bar, 50 μm. E – G Representative western blot ( E ) and graphical representations of ( F ) PGC-1α and ( G ) TOMM20 protein expression levels are shown. * P < 0.05 versus sham controls ( n = 5); †† P < 0.01, ††† P < 0.001 versus pcDNA group ( n = 5). H Representative micrographs show renal tubular morphologic injury, the expression of KIM-1 and caspase-3, and TUNEL assay in different groups, as indicated. Paraffin sections were subjected to periodic acid–Schiff (PAS) staining, stained with an antibody against KIM-1 and caspase-3. Frozen kidney sections were subjected to TUNEL staining. Arrows indicate positive staining. Scale bar, 50 μm. I – M Representative western blot ( I ) and graphical representations of ( J ) KIM-1, ( K ) FAS-L, ( L ) Bax and ( M ) cleaved caspase-3 protein expression levels are shown. * P < 0.05, ** P < 0.01, *** P < 0.001 versus sham controls ( n = 5); † P < 0.05, †† P < 0.01 versus pcDNA group ( n = 5). N Tubular injury score depending on PAS staining in three groups, as indicated. *** P < 0.001 versus sham controls ( n = 5); ††† P < 0.001 versus pcDNA group ( n = 5).
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Male mice were more susceptive to rhabdomyolysis-induced AKI and tubular apoptosis in kidney. A Experimental design. Female and male mice were intramuscularly injected with 50% glycerol at the dose of 7.5 ml/kg or normal saline respectively. Mice were euthanized 3 days after intramuscular injection. B Scr levels in four groups, as indicated. Scr was expressed as milligrams per deciliter. ** P < 0.01 versus sham controls in male group ( n = 5); †† P < 0.01 versus sham controls in female group ( n = 5); # P < 0.05 versus male group in glycerol group ( n = 5). C Graphical representations show three day-mortality in different genders after glycerol administration, as indicated. * P < 0.05 versus male group (n of male group = 20; n of female group = 17). D Representative micrographs show renal tubular morphologic injury and the expression of KIM-1 in different groups, as indicated. Paraffin sections were subjected to PAS staining and stained with an antibody against KIM-1. Arrows indicate positive staining. Scale bar, 50 μm. E Tubular injury score depending on PAS staining in four groups, as indicated. *** P < 0.001 versus sham controls in male group ( n = 5); ††† P < 0.001 versus sham controls in female group ( n = 5). F – J Representative western blot ( F ) and graphical representations of ( G ) KIM-1, ( H ) FAS-L, ( I ) Bax and ( J ) cleaved caspase-3 protein expression levels are shown. * P < 0.05, ** P < 0.01, *** P < 0.001 versus female group ( n = 5). K Representative micrographs show the expression of caspase-3 and TUNEL staining in different groups, as indicated. Paraffin sections were stained with an antibody against caspase-3. Frozen kidney sections were subjected to TUNEL staining. Arrow indicates positive staining. Scale bar, 50 μm.
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Growth layer groups (GLGs) in the thin section of a tooth. One GLG consists of an opaque layer and a translucent layer. The arrow represents the neonatal line. Scale bar = 200 μm.
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The expression of Sirtuin 6 was the key regulator for gender differences in rhabdomyolysis-induced AKI. A , B Representative western blot ( A ) and graphical representations of ( B ) Sirtuin 1-7 protein expression levels are shown. * P < 0.05, ** P < 0.01, *** P < 0.001 versus female group ( n = 5). C Representative micrographs show the expression of Sirtuin 6 in different groups, as indicated. Paraffin-embedded kidney sections were stained with an antibody against Sirtuin 6. Arrows indicate positive staining. Scale bar, 50 μm. D Representative micrographs showing the expression of PGC-1α and TOMM20 in different groups, as indicated. Frozen kidney sections were stained with an antibody against PGC-1α and TOMM20. Arrows indicate positive staining. Scale bar, 50 μm. E – G Representative western blot ( E ) and graphical representations of ( F ) PGC-1α and ( G ) TOMM20 protein expression levels are shown. * P < 0.05, *** P < 0.001 versus male group ( n = 5).
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Sirtuin 6 is the possible contributor to gender differences upon IRI. A Graphical representations show the relative abundance of Sirtuin 1-7 mRNA in different groups. ** P < 0.01, *** P < 0.001 versus sham controls in male group ( n = 5); † P < 0.05 versus sham controls in female group ( n = 5). B , C Representative western blot ( B ) and graphical representations of ( C ) Sirtuin 1-7 protein expression levels are shown. * P < 0.05, ** P < 0.01, *** P < 0.001 versus male group ( n = 5). D Representative micrographs showing the expression of Sirtuin 6 in different groups, as indicated. Paraffin-embedded kidney sections were stained with an antibody against Sirtuin 6. Arrows indicate positive staining. Scale bar, 50 μm. E Representative micrographs showing the expression of PGC-1α and TOMM20 in different groups, as indicated. Frozen kidney sections were stained with an antibody against PGC-1α and TOMM20. Arrows indicate positive staining. Scale bar, 50 μm. F – H Representative western blot ( F ) and graphical representations of ( G ) PGC-1α and ( H ) TOMM20 protein expression levels are shown. *** P < 0.001 versus male group ( n = 5).
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Male mice were more susceptive to IRI and tubular apoptosis in kidney. A Experimental design. Female and male mice were subjected to IRI or sham respectively, and euthanized 24 h after IRI. B Scr levels in four groups, as indicated. Scr was expressed as milligrams per deciliter. ** P < 0.01 versus sham controls in male group ( n = 5). C BUN levels in four groups, as indicated. BUN was expressed as milligrams per deciliter. * P < 0.05 versus sham controls in male group ( n = 5). D Representative micrographs show renal tubular morphologic injury, the expression of KIM-1 and caspase-3, and TUNEL assay in different groups, as indicated. Paraffin sections were subjected to periodic acid–Schiff (PAS) staining, stained with an antibody against KIM-1 and caspase-3. Frozen kidney sections were subjected to TUNEL assay. Arrows indicate positive staining. Scale bar, 50 μm. E – I Representative western blot ( E ) and graphical representations of ( F ) FAS-L, ( G ) Bax, ( H ) cleaved caspase-3 and ( I ) KIM-1 protein expression levels are shown. * P < 0.05, ** P < 0.01 versus male group ( n = 5).
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Histological sections of testes from birth to adulthood. LC: Leydig cells; SC: Sertoli cells; PG: Primordial germ cells; SG: Spermatogonia; PS: Primary spermatocytes; SS: Secondary spermatocytes; SPZ: Spermatozoa. (A) 0.1yr. (B) 2.5yr. (C) 3yr. (D) 3.5yr. (E) 4.5yr. (F) 6yr. (G) 8yr. (H) 13yr. Scale bar = 50 μm.
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Histological analysis of dorsolateral prostate in male aged rats treated with BPA for 3 months. The glandular cavity was slightly enlarged and increased in BPA-treated groups. ( a – d ) Representative sections of comparable regions were shown for vehicle control rats ( a ), and animals exposed to BPA (10 μg/kg/day) ( b ), BPA (30 μg/kg/day) ( c ), and BPA (90 μg/kg/day), ( d ) (scale bar: 50 μm, x40).
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AR increases acetylation of PGC-1α by downregulating Sirtuin 6 expression. A – C Representative western blot ( A ) and graphical representations of ( B ) Sirtuin 1-7 and ( C ) PGC-1α protein expression levels are shown. * P < 0.05, ** P < 0.01 versus control group ( n = 3). HKC-8 cells were transfected with pcDNA3 or AR overexpression plasmid for 24 h. D Representative ChIP assay results showing the binding of AR to the Sirtuin 6 gene promoter region. HKC‐8 cells were incubated with DHT (10 μMol/L) or not for 24 h. Cell lysates were precipitated with an antibody against AR, histone H3, or nonimmune IgG, and the ChIP assay was performed for Sirtuin 6 gene promoters. Total diluted lysate was used as the total genomic input DNA. E Graphical representations show the relative abundance of Sirtuin 1-7 mRNA in different groups. ** P < 0.01 versus control group ( n = 3). F – I Representative western blot ( F ) and graphical representations of ( G ) Sirtuin 1-7, ( H ) PGC-1α and ( I ) AR protein expression levels are shown. HKC-8 cells were incubated with DHT (10 μMol/L) and transfected with AR-shRNA for 24 h. * P < 0.05, ** P < 0.01, *** P < 0.001 ( n = 3); † P < 0.05, †† P < 0.01, ††† P < 0.001 ( n = 3). J Representative graphs show the binding of PGC-1α with Sirtuin 6 or acetyl. HKC-8 cells were transfected with pcDNA3 or AR overexpression plasmid for 24 h. K Representative graphs show the binding of PGC-1α with acetyl, and the expression of PGC-1α in different groups, as indicated. HKC-8 cells were treated with DHT (10 μMol/L) and transfected with Sirtuin 6 overexpression plasmid for 24 h. L Representative graphs show the binding of PGC-1α with acetyl, and the protein levels of AR in nuclear fractions in different groups, as indicated. HKC-8 cells were treated with DHT (10 μMol/L) for 24 h. M Representative graphs show the binding of PGC-1α with acetyl, and the protein levels of AR in nuclear fractions in sham control and IRI group in male mice, as indicated.
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Clustering analysis and scatter plot of microarray data. ( a ) Clustering analysis; ( b ) scatter plot; Group1: dorsolateral prostate of the BPA group; Group control: dorsolateral prostate of the control group. BPA: bisphenol A.
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