Tris tricine ready gel

Collecting cells and using the radio immunoprecipitation assay (RIPA) buffer (P0013B, Beyotime, Shanghai, China) in the presence of a phenylmethyl sulfonylfluoride (#8553, CST). Protein concentration were determined by the BCA Protein Assay Kit (#P0009, Beyotime, China). Equivalent amounts of the proteins (40 μg/line) were separated on Tris-Tricine Ready Gel (#1611210, Bio-Rad, Hercules, Calif, USA) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for nitrocellulose membrane polyvinylidene fluoride (PVDF) (ISEQ00010, Merck Millipore, Massachusetts, USA) blotting. The blotted membranes were blocked with 5% skim milk (#P0216-300g, Beyotime, Shanghai, China) for 1 hour at ambient temperature and incubated with primary antibodies overnight at 4^◦C. They were washed with tris-buffered saline Tween-20 (TBST) (CW0043S, CWBIO, Jiangsu, China), and the immunoreactive bands were visualized by enhanced chemiluminescence reagent. All bands were analyzed by NIH image 1.62. All experiments were conducted in triplicate.

Cells were collected from cultured dishes and lysed in lysis buffer. The main components of the lysis buffer were 20 mM Tris-HCl pH 7.6, 1 mM EDTA, 140 mM NaCl, 1% NP-40, 1% aprotinin, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM sodium vanadate. Protein concentration was determined using a BCA Protein Assay Kit (Pierce). Protein samples (40 μg of protein per cell line) were separated on a 5 to 20% Tris-Tricine Ready Gel (Bio-Rad) by SDS-PAGE for nitrocellulose membrane blotting. The blotted membrane was blocked with 5% skim milk for 1 h at room temperature and incubated overnight at 4 °C with primary antibody. The immunoreactive band was observed by enhanced chemiluminescence using horseradish peroxidase-conjugated IgG secondary antibodies. Band density was measured by densitometry, quantified using NIH image 1.62 gel map macros, and normalized to the specified sample in the same membrane.