Anti-CYP1B1 Antibody Picoband®

Western blot analysis of CYP1B1 using anti-CYP1B1 antibody (PB9546).
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 30 ug of sample under reducing conditions.
Lane 1: human MCF-7 whole cell lysates,
Lane 2: human PC-3 whole cell lysates,
Lane 3: rat heart tissue lysates,
Lane 4: mouse heart tissue lysates.
After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA 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-CYP1B1 antigen affinity purified polyclonal antibody (Catalog # PB9546) at 0.5 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 CYP1B1 at approximately 61 kDa. The expected band size for CYP1B1 is at 61 kDa.

aHSC release LTB4R2 ligands in a manner dependent on CYP1B1. a TEAD-luciferase activity in Huh7 cells treated with conditioned medium (CM) from LX2 cells transduced with scrambled shRNA (SCRsh-CM) vs. SCD-shRNA (SCDsh-CM) as compared to the media without Huh7 cells (Media). * p < 0.05 by two-sided t-test. Data presented as means ± SEM ( n = 4 separate experiments). b TEAD-luciferase activity in Huh7 cells exposed to SCRsh-CM vs. SCDsh-CM in the presence of the TBXAS1 inhibitor Ozagarel or vehicle DMSO. * p < 0.001 vs. Media, # p < 0.001 vs. DMSO by two-sided t-test. Data presented as means ± SEM ( n = 3 experiments). c A scRNA - seq t-SNE plot showing Cyp1b1 + cells and violin plots revealing selective Cyp1b1 expression by Fbln2 + cells. Cell numbers for different cell type groups are provided in Supplementary Data . d Contour FACS plots of liver mesenchymal cells isolated from control vs. DEN + WAD treated Rosa26mTmG;Col1a1-Cre; ( mTmG;CC ) mouse, gated by DAPI ( Y -axis) for Vit A fluorescence and FITC (X-axis) for Col1a1-GFP, revealing VitA + GFP - quiescent HSC (blue), VitA + GFP + aHSC (red), and VitA − GFP + cells (green). FACS gating strategies are provided in . e scRNA-seq analysis for expression of 12-HHTrE biosynthetic genes in VitA + GFP + (top) and VitA − GFP + (bottom) subpopulations from DEN+WAD mouse (DEN) vs. normal (Cont.) livers. f Violin plots for Cyp1b1 expression by subpopulations based on Lrat , Thy1 , and Fbln2 expression in VitA + GFP + cells from the DEN mouse and g in VitA − GFP + cells. (See Supplementary Data for parameter values for violin plots and cell numbers for different subpopulations). h CRISPR/Cas9 ablation of CYP1B1 in LX2 cells using the guide RNA-A (sgRNA-A) or -B (sgRNA-B) (top), represses CYP1B1 mRNA. * p < 0.05 vs. control by two-sided t-test. Data presented as means ± SEM ( n = 3 separate samples). i Oxylipin concentrations in CM from LX2 cells with CYP1B1 KD are described above. * p < 0.05 vs. control by two-sided t-test. Data presented as means ± SEM ( n = 4 separate samples). (Raw data provided in Supplementary Data in Supplementary File). j Reduced stimulatory effects of CM from CYP1B1 ablated LX2 cells on the TEAD promoter activity in Huh7 cells (right). *** p < 0.001 vs. Control CM by two-sided t-test. Data presented as means ± SEM ( n = 3 experiments). For all relevant figures, source data and exact p values are provided in the file.
Index in PubMed under a CC BY license. PMID: 37156770

HCC growth is dependent on LTB4R2. a IB analysis of LTB4R2 and NaKATPase (membrane), pYAP1, pGSK3β, GSK3β, HuR, GAPDH (cytosolic), YAP1 and CTNNB1 (nuclear) proteins from B6 mice subjected to the DEN + WAD regimen and injected with AAV vector (4 × 10 11 GC per mouse) expressing SCR-shRNA (SCR-sh) vs. LTB4R2-shRNA (LTB4R2-sh) one month prior to the end of experiment ( n = 6 mice per group). b Liver tumor development in the mice with SCR-sh vs. LTB4R2-sh treatment depicted by representative images and total tumor volume and multiplicity. * p < 0.05 vs. SCR-sh mice by two-sided t-test. Data presented as means ± SEM ( n = 9 mice each). c qPCR data for oxylipin synthetic genes, LTB4R2 , and YAP1 in patient HCC ( n = 6) vs. normal subject livers ( n = 6). * p < 0.05, ** p < 0.01 vs. normal liver by two-sided t-test. Data presented as means ± SEM. d Representative IHC-HRP staining for CYP1B1 and LTB4R2 of patient HCC liver sections (×200) from four patient samples examined. Areas demarked by broken lines are HCC. e Top: Growth of patient HCC organoid (model-1 and model-2) in the presence of DMSO (vehicle) or the LTB4R2 antagonist LY255283. * p < 0.05, ** p < 0.01, *** p < 0.005 vs. DMSO by two-way ANOVA with post hoc test. Data presented as means ± SEM ( n = 3 experiments). Bottom: Growth of patient HCC organoid model-1 and model-2 without (CTRL) or with infection with adenovirus expressing scrambled shRNA ( shCTRL ) or LTB4R2 shRNA (shLTB4R2). * p < 0.05, *** p < 0.005 vs. DMSO ( n = 3 experiments). f Schematic diagram of HCC promotion initiated by SCD-CYP1B1-dependent release of 12-HHTrE by Lart + Fbln2 + aHSC, activating LTB4R2-CTNNB1-YAP1 pathway in HCC cell. For all relevant figures , source data and exact p values are provided in the file.
Index in PubMed under a CC BY license. PMID: 37156770

IHC analysis of CYP1B1 using anti-CYP1B1 antibody (PB9546).
CYP1B1 was detected in paraffin-embedded section of Mouse Kidney Tissue. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-CYP1B1 Antibody (PB9546) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

IHC analysis of CYP1B1 using anti-CYP1B1 antibody (PB9546).
CYP1B1 was detected in paraffin-embedded section of Rat Kidney Tissue. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-CYP1B1 Antibody (PB9546) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

IHC analysis of CYP1B1 using anti-CYP1B1 antibody (PB9546).
CYP1B1 was detected in paraffin-embedded section of Human Liver Cancer Tissue. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-CYP1B1 Antibody (PB9546) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

Flow Cytometry analysis of SiHa cells using anti-CYP1B1 antibody (PB9546).
Overlay histogram showing SiHa cells stained with PB9546 (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-CYP1B1 Antibody (PB9546,1μg/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.