Anti β actin

Protein isolation and Western blotting were performed according to standard procedures. For protein isolation from cells, the cells were washed with ice cold phosphate buffered saline (PBS) and lysed in radioimmunoprecipitation assay (RIPA) buffer containing phosphatase and protease inhibitors (Roche). For mouse and human biopsies, the samples were dissociated in RIPA buffer using a bead-beater and metal beads. All samples were then sonicated for 30 seconds and centrifuged (10 minutes at 12,000 g at 4 °C), and the supernatant was transferred into fresh tubes. Protein concentration was detected using a BCA assay (Thermo Fisher Scientific). For Western blots, aliquots with equal amounts of protein were separated by electrophoresis on polyacrylamide gels, and the proteins blotted on nitrocellulose membranes. The membranes were then blocked in blocking buffer (3% milk, 1% bovine serum albumin [BSA] in tris-buffered saline with 0.5% Tween) for 1 hour and incubated overnight at 4 °C with anti-ACE2 (Clone E-11, Santa Cruz Biotechnology), anti-phospho-STAT1 (Tyr701, clone 58D6; Cell Signaling Technologies), anti-STAT1 (clone 42H3; Cell Signalling Technologies), or anti-β-actin (Clone AC-74, Sigma-Aldrich) antibodies. On the next day, the membranes were washed in tris-buffered saline with 0.5% Tween, incubated with horseradish peroxidase (HRP)-coupled secondary antibodies (Jackson Immunolabs), washed again, and immunoreactive proteins detected using ELC substrate (Thermo Fisher Scientific) and X-ray films (GE Healthcare).

To prepare protein-cell lysates for IP, cells were solubilized in CelLytic M buffer (Sigma-Aldrich, Saint Louis, MO, USA). Protein-cell lysates were prepared as previously described [12 (link),14 (link)]. Western blot analysis was performed as in previous studies [10 (link),11 (link),12 (link),13 (link),14 (link)]. In this study, experimental cell-protein extraction and lysis were carried out using RIPA (Radio-Immunoprecipitation Assay) buffer supplemented with PMSF and a protein inhibitor. Subsequently, protein concentrations were determined, and the lysates were heated with a loading buffer at 100 °C for 10 min. Thirty micrograms of proteins from each sample were loaded onto 10%. SDS-PAGE gels, transferred to PVDF membranes, blocked with 5% BSA, and then exposed to the relevant antibody. Western blots (wet Western blots) were performed. Membranes (PVDF, Thermofisher, Waltham, MA, USA; TS-88518) were blocked and probed with the appropriate primary antibodies. These are anti-FLAG (F3165; Sigma-Aldrich, Saint Louis, MO, USA), anti-β-actin (A5441; Sigma-Aldrich, Saint Louis, MO, USA), anti-β-catenin (C2206), anti-PAR₂ (AB180953; Abcam, Cambridge, UK and SC-13,504 Santa Cruz Biotechnology, Dallas, TX, USA), anti-PAR₄ (AB5787; Abcam (Cambridge, UK): SC-13504 Santa Cruz Biotechnology (Dallas, TX, USA)), anti-HA (901503; Biolegend, San Diego, CA, USA), anti-LRP6 (BS-7007R, Bioss Antibodies, Woburn, MA, USA), anti-phospho-LRP6 (Cell Signaling Technology, Danvers, MA, USA), anti-AKT (AB8805; Abcam, Cambridge, UK), anti-p53 (AB17990; Abcam, Cambridge, UK), anti-p21 (AB109520; Abcam, Cambridge, UK), and anti-GAPDH (AB9485; Abcam, Cambridge, UK). These antibodies were suspended in 3% BSA (#A5611, Sigma-Aldrich, Head office Kanagawa, Japan) in 10 mM Tris-HCl (T1503; Sigma-Aldrich, Saint Louis, MO, USA), pH 7.5, 100 mM NaCl, and 0.1% Tween-20 (P9416; Sigma-Aldrich, Saint Louis, MO, USA). After extensive washes, blots were incubated with secondary antibodies conjugated to horseradish peroxidase (HRP) anti-mouse (#ab6728; Abcam, Cambridge, UK) or anti-Rabbit (Abcam, Cambridge, UK; #ab6721). Immunoreactive bands were detected by the enhanced chemiluminescence (ECL) reagent (Pierce, Rockford, IL, USA).

Whole-cell lysates collected from 100,000 cells were used per lane. Antibodies used were: anti-AR (N-20, Santa Cruz; Santa Cruz, CA); anti-Beta Actin (Sigma-Aldrich); anti-ΔNp63 (4A4, Santa Cruz); anti-Sox2 (D6D9 XP, Cell Signaling Technology; Beverly, MA); anti-Nanog (D73G4 XP, Cell Signaling Technology); anti-Oct4 (C30A3, Cell Signaling Technology); and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Cell Signaling Technology). Secondary horseradish peroxidase-conjugated antibodies were from Cell Signaling Technologies, and HRP detected using SuperSignal West Femto Chemiluminescent Substrate (Peirce/Thermo Scientific; Rockford, IL). Alternatively, secondary antibodies from Rockland (Gilbertsville, PA) were used and data captured using a Licor Odyssey system (Lincoln, NE).
Immunostaining for Sox2 (D6D9 XP, Cell Signaling Technology; rabbit monoclonal) was performed on formalin-fixed, paraffin-embedded (FFPE) sections managed either by the University of Chicago Human Tissue Resource Core facility or the Northwestern University/University of Chicago Prostate SPORE program and their Specimen Procurement Program. After deparaffinization and rehydration, tissues were treated with antigen retrieval buffer (S1699 from DAKO; Glostrup, Denmark) in a steamer for 20 minutes. Anti-Sox2 antibody (1∶25 dilution) was applied for 1 hour at room temperature in a humidity chamber. Following TBS wash, the antigen-antibody binding was detected with Envision+system (DAKO, K4001 for mouse primary antibodies) and DAB+Chromogen (DAKO, K3468). Tissue sections were briefly immersed in hematoxylin for counterstaining and were cover-slipped. Tissues were analyzed by a trained Genitourinary Pathologist and scored on percentage of cells with positive nuclear staining (0 = no staining; 1 = 1–10% positive cells; 2 = 11–50% positive cells; and 3 = >50% positive cells); as well as the intensity of staining (0 = no staining; 1 = weak staining; 2 = moderate staining; 3 = strong staining). For images, slides were digitized using a Pannoramic Scan Whole Slide Scanner (Cambridge Research and Instrumentation; Hopkinton, MA) and images captured using the Pannoramic Viewer software version 1.14.50 (3DHistech; Budapest, Hungary).
For immunofluorescence, tissues were deparaffinized, rehydrated, and treated with antigen retrieval buffer. Antibody binding of Sox2 (D6D9, Cell Signaling Technology; Alexa-Fluor 555-conjugated, 1∶50 dilution in TBST) and p63 (4A4, Santa Cruz Biotechnology; Alexa-Fluor 647 conjugated, 1∶50 dilution in TBST) were conducted for 1 hr at room temperature. Tissues were counter-stained with DAPI and mounted using Fluoromount-G (Southern Biotech, Birmingham, AL). Fluorescent images were captured using a Leica TCS SP2 AOBS Laser Scanning Confocal microscope.