Skin tissues were homogenized with CelLyticTM MT Cell Lysis Reagent (Sigma-Aldrich). Ten micrograms of the protein lysate was resolved by SDS-PAGE, transferred to a PVDF membrane, and probed with antibodies against PCNA (1:1,000; ab29, Abcam), Cyp1a1 (1:1,000; #871, Daiichi Pure Chemical Co., Ltd., Tokyo, Japan), Cyp1b1 (1:1,000; ab185954, Abcam, Cambridge, UK), Actin (1:1,000; sc1616-HRP, Santa Cruz Biotechnology, Santa Cruz, CA), Pan-Ras (1:1000; Component of STA-440, Cell Biolabs, Inc., San Diego, CA), EGF receptor (1:1000; #2232, Cell Signaling Technology, Danvers, MA), phosphorylated (Y1086) EGF receptor (1:1,000; ab32086, Abcam), MEK1 (1:1,000; ab32091, Abcam), phosphorylated (E342) MEK1 (1:1,000; ab396379, Abcam), Cleaved Caspase-3 (1:5000; #9661, Cell Signaling Technology), Flag-M2 (1:1000; F3165, Sigma-Aldrich), and p16INK4a (1:500; sc1207, Santa Cruz Biotechnology). Specific antigen-antibody complexes were visualized using horseradish peroxidase-conjugated secondary antibodies and Chemi-Lumi One (Nacalai Tesque) or ImmunoStar reagent (Wako, Osaka, Japan).
Ab185954
Manufactured by Abcam
Sourced in United Kingdom
Ab185954 is a primary antibody product offered by Abcam. It is designed for use in various laboratory techniques, including immunohistochemistry, immunocytochemistry, and Western blotting. The core function of this product is to aid in the detection and identification of its target antigen.
Automatically generated - may contain errors
Lab products found in correlation
Ultravision detection system, by Thermo Fisher Scientific (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Cellytic mt cell lysis reagent, by Merck Group (1 mentions)
F3165, by Merck Group (1 mentions)
Ab32091, by Abcam (1 mentions)
Chemi lumi one, by Nacalai Tesque (1 mentions)
Sc 1616 hrp, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Gel imaging system, by Bio-Rad (1 mentions)
Ab7090, by Abcam (1 mentions)
Ab262696, by Abcam (1 mentions)
Ab253197, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Ab32572, by Abcam (1 mentions)
Dulbecco s modified eagle s medium f12 dmem f12, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Immun blot pvdf membrane, by Bio-Rad (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Resveratrol, by Merck Group (1 mentions)
5 protocols using ab185954
1
Skin Tissue Protein Immunoblotting
2
Western Blot Analysis of Key Proteins
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The proteins were isolated with RIPA, which were further transferred to a PVDF membrane (Millipore, Billerica, MA, USA). Then, the membranes were incubated with antibodies against MeCP2 (Abcam, 1:1,000, ab253197), CYP1B1 (Abcam, 1:1,000, ab185954), Wnt4 (Abcam, 1:1,000, ab262696), β‐catenin (Abcam, 1:1,000, ab32572), and β‐actin antibody (Abcam, 1:10,000, ab8226) overnight, then hybridized with a secondary antibody (Abcam, 1:10,000, ab7090) for 60 min. Blots were visualized by the GEL imaging system (Bio‐Rad) and subsequently analyzed with ImageJ software.
3
Immunostaining of CYP1B1 in BPH Tissue
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Immunostaining of CYP1B1 was performed on specimens of BPH. Slides consisting of 4 μm slices of tissue underwent the protocol of the UltraVision Detection System (Thermo Fisher Scientific, Waltham, MA) according to manufacturer's instructions. After 12‐hour incubation with rabbit monoclonal antibody for CYP1B1 (1:500 dilution, #ab185954, Abcam, Cambridge, UK), 3, 3′‐diaminobenzidine (DAB) was added as chromogen followed by counterstaining with haematoxylin. Cellular expression levels were analysed by the intensity of positive cells using ImageJ software (http://rsb.info.nih.gov/ij ) and ranked on an overall scale from 0 to 3, with 0 indicating the absence of staining; 1, weak staining; 2, moderate staining; and 3, strong staining.
4
Resveratrol Modulates CYP1B1 Expression
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Resveratrol was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). 15-HETE-D8 was purchased from Cayman Chemical (Ann Arbor, MI, USA). Dulbecco’s Modified Eagle’s Medium/F-12 (DMEM/F-12) and DMEM were obtained from Gibco, Life Technologies (Grand Island, NY, USA). Fetal bovine serum was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). TRIzol reagent was purchased from Invitrogen Co. (Carlsbad, CA, USA). High-Capacity cDNA Reverse Transcription kit and SYBR® Green PCR Master Mix were purchased from Applied Biosystems (Foster City, CA, USA). Real-time PCR primers were prepared by Integrated DNA Technologies (Coralville, IA, USA). Immun-Blot® PVDF membrane was purchased from Bio-Rad Laboratories (Hercules, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse monoclonal was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Recombinant monoclonal anti-CYP1B1 antibody [ab185954] was purchased from abcam (Toronto, ON). Chemiluminescence Western blotting detection reagents (ECL) were obtained from GE Healthcare Life Sciences (Pittsburgh, PA, USA). All of the other chemicals used were obtained from Fisher Scientific Co. (Toronto, ON).
5
COMT Expression in Prostate Pathologies
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Immunostaining of COMT was performed on specimens of BPH and prostate cancer (patient characteristics in Table 1 ). Slides consisting of tissue sections underwent the protocol of the UltraVision Detection System (Thermo Fisher Scientific, Waltham, MA) according to manufacturer’s instructions. After 12‐hour incubation with rabbit monoclonal antibody for COMT (1:500 dilution, #ab185954, Abcam, Cambridge, MA), 3, 3′‐diaminobenzidine (DAB) was added as chromogen followed by counterstaining with hematoxylin. Cellular expression levels were analyzed by the intensity of positive cells using Fiji ImageJ [21 (link)]. The IHC images of normal prostate tissues were obtained from the Protein Atlas database (http://www.proteinatlas.org ) [22 (link)].
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