Ripa protein extraction buffer

The total protein of the cells was extracted using RIPA protein extraction buffer (Beyotime, Shanghai, China) containing protease inhibitor. Proteins were separated using SDS-PAGE gels and transferred to a polyvinylidene fluoride (PVDF) membrane using a iBlot 2 Dry Blotting System (Life technologies, Thermo Fisher Scientific, Waltham, MA). The non-specific reactivity was blocked with nonfat milk at 4°C for one hour. The PVDF membranes were then incubated with primary antibodies including anti-decorin (1:1000), anti-E-cadherin (1:10000), anti-fibronectin (1:1000), anti-vimentin (1:1000), anti-Snail (1:1000), anti-Slug (1:1000), anti-Twist (1:1000), anti-LC3B (1:2000), anti-p62 (1:10000), anti-c-Met (1:1000), anti-p-c-Met (1:1000), anti-Akt (1:1000), anti-p-Akt (1:1000), anti-mTOR (1:10000), anti-p-mTOR, anti-ERK1/2 (1:10000), anti-p-ERK1/2, anti-β-actin (1:5000) and anti-GAPDH (1:5000) at 4°C overnight. These antibodies were from Abcam (Cambridge, MA). The membranes were then washed in TBST and incubated with Horseradish peroxidase-conjugated secondary antibodies (Beyotime, Shanghai, China) for 1 h. A Super ECL Plus Detection reagent (Applygen Technologies, Beijing, China) was used to develop the bands, which were captured by a Tanon-4200 Gel Imaging System (Tanon, Shanghai, China).

Western blot was performed as previously described (Zhang et al., 2019a (link)). Briefly, HepG2 cells were seeded in 60 mm culture dishes (5 × 10⁵ cells) for 24 h. After the treatments for 24 h followed by insulin (100 nM) incubation for 20 min, cells were washed with cold PBS, and then lysed with cold Radio Immunoprecipitation Assay (RIPA) protein extraction buffer containing 1% protease and phosphatase inhibitors (Beyotime biotechnology, Beijing, China) for 30 min on ice. An aliquot of 20 μg of the supernatant protein from each sample was heated with 4 × sodium dodecyl sulfate (SDS) sample buffer at 95 °C for 8 min, and separated electrophoretically on a 10% SDS–polyacrylamide gel. Subsequently, proteins were transferred onto PMSF membranes for 3 h and blocked for 1 h. PMSF membranes were then exposed to indicate primary antibodies in blocking buffer at 1:1,000 dilutions overnight at 4°C. The membranes were then incubated with the anti-rabbit IgG secondary antibody or and anti-mouse IgG HRP-linked antibody at 1:2,000 dilutions for 1 h. Visualization was performed using Tanon 5200 Multi chemiluminescent imaging system (Tanon Science & Technology Co., Ltd., Shanghai, China) with enhanced-chemiluminescence substrate, and the blots were analyzed using Image J software. Protein levels were corrected with values determined on β-actin blots. Three replicates were used.

Western-blotting was implemented as the procedures reported by Fathi et al.^{24 (link)}. We extracted protein from each group of A549 and NCI-H23 cells by using a RIPA protein extraction buffer (Beyotime, Shanghai, China). By using the BCA protein test kit (Beyotime, Shanghai, China), we detected protein concentrations. And then, we separated and electrotransferred equal amounts of protein (40 μg) from each sample onto polyvinylidene difluoride (PVDF) membranes. After blocked and incubated with the primary antibodies, the membranes were then incubated with secondary antibodies. In the experiment, we used the antibodies as follows: anti-SLC7A11 antibody (1:1500, ab175186, Abcam), anti-GPX4 (1:2000, ab125066, Abcam), anti-β-actin antibody (1:1000, ab8227, Abcam). Finally, detection was done by chemiluminescence (Millipore Corporation, Temecula, CA, USA).