Phenylmethylsulphonyl fluoride

Cells were all harvested with cell scrapers after washing 3 times with pre-cold PBS. Then, the harvested cells were centrifuged for 3 minutes at 1000x g, and the pellets were saved. The cell pellets were lysed with RIPA lysis buffer (KeyGen Biotech, Shanghai, China) containing 1 mmol/l phenylmethylsulphonyl fluoride (KeyGen Biotech, Shanghai, China) and 1 mmol/l phosphatase inhibitor cocktail (KeyGen Biotech, Shanghai, China). The mixture was centrifuged for 10 minutes at 12000x g, and the supernatant containing proteins was saved.

Western blotting was performed following the official protocol. In brief, protein was extracted from the cells and tissues via treatment with 1× radioimmunoprecipitation assay buffer (KeyGEN, Nanjing, China) containing 1% phenylmethylsulphonyl fluoride (KeyGEN). The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA), blocked with 5% skim milk for 1 h at room temperature, immunoblotted overnight with primary antibodies and for 1.5 h with secondary antibodies, and visualized using an Odyssey CLx Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA). Antibodies against TIGD1 were purchased from Wanlei Technology, and antibodies against β-actin were purchased from Proteintech.