Bicinchoninic acid assay kit

The detailed information for primary antibodies used in this study is described in Supplementary Table S4. For immunoblotting analyses, cells or tissue samples were lysed in RIPA buffer with 1 × protease inhibitors and phosphatase inhibitors (Bimake, #B14002 and #B15003, respectively). Quantification of protein concentrations was performed using the bicinchoninic acid assay kit (Yeasen, # 20201ES90). Equal amounts of protein extracts were separated by SDS-PAGE and transferred onto PVDF membranes (Millipore, #IPVH00010). The membranes were incubated with indicated primary antibodies and detected with Super ECL detection kit (Yeasen, # 36208ES76). Quantitation of immunoblotting bands was performed using ImageJ software, and expression levels of target proteins were normalized to those of internal control Vinculin.

Total protein was extracted using radioimmunoprecipitation assay buffer (Beyotime, China) with inhibitor cocktail, and the concentrations were quantified by bicinchoninic acid assay kit (Yeasen, China). Western blotting was done according to standard protocols. Antibodies against TICRR (1:1000, NBP2-41283), PPIF (1:1000, 18466-1-AP), β-tubulin (1:5000, 30303ES10), GAPDH (1:5000, 30203ES10) were used as primary antibodies. The immunoblot bands intensity were quantified with ImageJ software (NIH, MD, USA).

The cells were lysed in Radio Immunoprecipitation Assay (RIPA) Lysis buffer supplemented with proteases (Yeason, China) and phosphatase inhibitors (Yeason, China) for 20-min incubation on ice. The bicinchoninic acid assay kit (Yeason, China) was conducted to measure the protein concentration in cell lysates. About 8%–12% SDS-polyacrylamide gels were used to separate total protein lysates from different samples, then Polyvinylidene fluoride (PVDF) membranes were electrophoretically transferred (130 V, 50 min at 4°C). The blocking solution was applied to PVDF membranes followed by incubation with specific antibodies overnight at 4°C. The following antibodies were used in this study: anti-β-actin (1:4000, ab8227, Abcam), anti-MMP9 (1:1000, CST, Massachusetts, United States), anti-MMP2 (1:1000, CST, Massachusetts, United States), anti-MEK (1:1000, CST, Massachusetts, United States), anti-p-MEK (1:1000, CST, Massachusetts, United States), anti-p-ERK (1:1000, CST, Massachusetts, United States), anti-ERK (1:1000, CST, Massachusetts, United States), anti-GPX4 (1:1000, CST, Massachusetts, United States).