Anti mouse hrp 7076

Western blot analysis was performed as described previously [48 (link)]. For whole-cell lysates, cells were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors (both from Roche, Mannheim, Germany). One representative experiment out of at least three performed is shown. Primary antibodies were purchased from Cell Signaling, Frankfurt, Germany [anti-MDM2 (86934; used for all western blots); anti-p21 (2947); anti-PML (33156; used for all western blots)], Santa Cruz Biotechnology [anti-p53 (DO-1, used for all western blots except for blots shown in Figs. 5B, 6B); anti-p53 (FL393, for Figs. 5B, 6B), BD Transduction Laboratories, Heidelberg, Germany [anti-Bax (610982)], Merck Millipore, Darmstadt, Germany [anti-DR4 (AB16955)], Epitomics, California, USA [α-Tubulin (1878-1)], ProScience Incorporated, Poway, USA [anti-TRAIL-R2 (2019)] and from Sigma-Aldrich [anti-β-Actin (A5441)]. Secondary antibodies were purchased from Cell Signaling [anti-rabbit-HRP (7074) and anti-mouse-HRP (7076)].

Protein extracts (20 µg) in RIPA buffer supplemented with antiprotease-antiphosphatase (Halt™ Protease and Phosphatase Inhibitor Single-Use Cocktail, Thermo Scientific) were reduced and size-separated on 4-15% Mini-PROTEAN^® TGX Stain-Free™ Precast Gels (Bio-Rad, Hercules, CA, United States) and transferred onto nitrocellulose membranes using an iBlot2™ Gel Transfer Device and iBlot2™ Nitrocellulose Regular Stacks (IB23001, Invitrogen). Membranes were incubated with specific primary antibodies followed by corresponding secondary-HRP antibodies. Mouse anti–β-actin antibody (A2228) (1/1,000) was from Sigma-Aldrich. Rabbit anti–phopho-mTOR (#2971, 1/1,000), rabbit anti-mTOR (#2983, 1/1,000), anti-mouse HRP (#7076, 1/10000), and anti-rabbit (#7074, (1/5,000) antibodies were from Cell Signaling Technology (Denver, CO, United States). Immunodetection was carried out using Clarity™ Western ECL Substrate (#170-5,061, Bio-Rad). Image acquisition was performed using Las-3000 (Fujifilm, Bussy-Saint-Georges, France). Densitometric quantification was performed using ImageJ software.

Protein was isolated from snap-frozen CU-PC01 tumours using RIPA buffer (Universal Biologicals Ltd., Cambridge, UK #39244.01) supplemented with a protease/phosphatase inhibitor cocktail (Cell Signalling Technology, Leiden, Netherlands, #5872S) and homogenised with 1.4 mm ceramic beads (VWR, Leicestershire, UK, #432-0372P) in a FastPrep24-5G homogeniser (MP Biomedicals, CA, USA, #116005500). Protein concentrations were determined using a Bradford assay (Bio-Rad, Watford, UK, #5000205), before equal amounts of protein were separated using 10% Mini-PROTEAN TGX gels (Bio-Rad, #4561034) and transferred onto mini PVDF membranes (Bio-Rad, #1704156) using the trans-blot turbo transfer system (Bio-Rad, #1704150). Membranes were blocked in 5% BSA (Sigma), incubated with the primary antibody overnight at 4 °C, the secondary antibody for 1 h at room temperature and protein detected using the Clarity ECL substrate (Bio-Rad, #1705061) and the ChemiDoc Imaging System (Bio-Rad). Primary antibodies included Cell Signalling Technology antibodies p-AKT T308 1:1000 (#13038), AKT 1:1000 (#9272) and GAPDH 1:2000 (#5174), and BD Biosciences antibody RB 1:1000 (#554136). Secondary anti-rabbit-HRP (#7074) and anti-mouse-HRP (#7076) antibodies were sourced from Cell Signalling Technology.