Western Blot Analysis of PHGDH Protein

Total cellular proteins were prepared and treated as described previously (Wang et al., 2017 (link)). Radio immunoprecipitation lysis buffer (Beyotime, Shanghai, China) with 1% phenylmethylsulfonyl fluoride (Beyotime, Shanghai, China) was used to extract total proteins from the cells. Proteins were separated by 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, MA). After blocking with 5% BSA, the membranes were incubated with primary antibodies for PHGDH (HPA021241; Sigma, MO) and β-actin (6008-1-Ig; Proteintech, Wuhan, China). Thereafter, an HRP-labeled anti-rabbit/mouse IgG (Beyotime, Shanghai, China) was added as the secondary antibody. The blots were visualized using ECL reagent (Thermo Scientific, MA) and exposed to a chemiluminescence detection system (Tanon, Shanghai, China). Image J software was used for data analysis.