Pmsf proteinase inhibitors cocktail

Cells were washed twice with cold PBS. Total protein was extracted by RIPA lysis buffer accompanied with 1 mM PMSF proteinase inhibitors cocktail (Beyotime, China) and phosphatase inhibitors cocktail (Yeasen, China) and then quantified using a BCA Protein Assay kit (Beyotime). Western blotting was performed as previously described^{29 (link)}. Protein extracts (20 μg) were separated by 10% SDS-PAGE and then transferred to 0.45 µm polyvinylidene fluoride (PVDF) membranes (Millipore). After blocking in 5% bovine serum albumin (BSA) for 1 h at room temperature, the membranes were incubated with specific primary antibodies (FOXO3: ProteinTech, 10849-1-AP; phosphor-FOXO3 (p-FOXO3): Cell Signaling Technology (CST), 5538; ERK: CST, 4695; phosphor-ERK (p-ERK): CST, 4377; AKT: CST, 4691; phosphor-AKT (p-AKT): CST, 9277; GAPDH: ProteinTech, 10494-1-AP) at 4 °C overnight. Then the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Neobioscience, China) for 1 h at room temperature. Protein bands were visualized by chemiluminescent ECL reagent (Millipore, Danvers, MA, USA) using Tanon 4600 Chemiluminescent Imaging System (Tanon, China). For each experiment, more than three biological repetitive tests were carried out.

Total protein was extracted by RIPA lysis buffer with 1 mM PMSF proteinase inhibitors cocktail (Beyotime, China) and quantified using Pierce BCA Protein Assay kit (Thermo Scientific, USA). Protein extracts (15 μg) were separated by electrophoresis through 10% SDS-PAGE and then electroblotted onto 0.22 μm PVDF membranes (Millipore, Germany) and blocked in 5% milk in Tris-buffered saline containing Tween-20 (TBST) for 1 h at room temperature. The membranes were incubated with primary antibodies, including anti-GPX4 (1:1000), anti-FTH1 (1:1000), anti-HIF-1α (1:1000), anti-HIF-2α (1:1000) and anti-β-actin (1:5000) overnight at 4 °C, and HRP-conjugated secondary antibodies (Neobioscience, China) for 1 h at room temperature. Protein signals were detected with an enhanced chemiluminescent kit (Thermo Scientific, USA).