Horseradish peroxidase conjugated sheep anti mouse and sheep anti rabbit antibodies

For immunoblotting 2.5 × 10⁵ to 1 × 10⁶ cells were lysed in laemmli buffer containing 5% 2-β-mercaptoethanol (Sigma Aldrich, Germany), sonicated and boiled for 5 min at 95 °C. Proteins were size fractionated on prestained gradient polyacrylamide gels (Mini-PROTEAN^®TGX Stain-Free™ Precast Gels, Biorad, Germany), blotted onto 0.2 μm PVDF membrane, blocked 2 h in 5% low fat milk powder and incubated overnight with primary antibodies against ITGAV (BD Biosciences, Germany), ITGA5 (Biolegend, USA), AKT, phAKT (S473), phERK (Thr202/Tyr204), ERK, PLIN1, FABP4, YAP, TAZ (all from Cell signaling, USA), p21^Cip1 (ThermoScientific, Austria), survivin (Enzo Lifesciences, Switzerland), p73 (Imgenex, USA), p53 (MyBiosource, USA), GAPDH (6c5,Santa Cruz, Germany). After washing, horseradish peroxidase-conjugated sheep-anti-mouse and sheep-anti-rabbit antibodies (all from Cell signaling, USA) were incubated for 1 h and the reaction was visualized by enhanced chemiluminescence reagent ECL (Biorad, Germany) using a Biorad ChemidocMP gel analyzer for detection. Quantification was performed with the ImageLab 5.0 software (Biorad, Germany) according to the manufacturer’s instructions. Total protein loading was visualized using the ChemidocMP gel analyzer and employed as an internal loading control for all immunoblots59 (link).

5×10⁴ to 2×10⁵ cells were directly lysed in Laemmli buffer containing 10% dithiothreidol (DTT, Sigma Aldrich, Germany), sonicated, and boiled for 5min at 75°C. Proteins were size fractioned on pre-stained gradient polyacrylamide gels (MiniPROTEAN®TGX Stain-Free™ Precast Gels, Biorad, Germany), blotted onto 0.2μm PVDF membrane, blocked for 2 h in 5% reconstituted low fat milk powder. Membranes were incubated overnight with primary antibodies against HK1, HK2, PFKP, PKM2, LDHA, FASN, ACC, ACSS2, ACLY, PLIN1, FABP4, CEBPA, CEBPB, PPARG, and GAPDH (all obtained from Cell Signaling, Germany) in Tween-TBS (TTBS) supplemented with 5% BSA. SREBP1 (clone 2A4) antibody was obtained from Novus Biological. After extensive washing, horseradish peroxidase-conjugated sheep-anti-mouse and sheep-anti-rabbit antibodies (Cell Signaling, Germany) were incubated for 1 h, and the reaction was visualized by enhanced chemiluminescence reagent ECL (BioRad, Germany) using a BioRad ChemidocMP gel analyzer for detection. Quantification was carried out using the ImageLab 5.0 software (Biorad, Germany) according to the manufacturer’s instructions, with normalization of protein expression to total protein loading.