Harvested cells were lysed using M‐PERTM Mammalian Protein Extraction Reagent (Thermo Scientific) with protease and phosphatase inhibitors (Beyotime). Protein concentration was determined by BCA protein quantification kit (Beyotime). Equal amounts of the proteins were loaded into SurePAGEᵀᴹ precast polyacrylamide gels with a gradient between 4% and 20% (GenScript) and transferred to PVDF membranes (Millipore) by eBlot® L1 wet protein transfer system (GenScript). After blocking, the membranes were incubated with primary antibodies overnight at 4℃. Then, the membranes were incubated with HRP‐conjugated anti‐mouse or anti‐rabbit secondary antibody (GAM0072 or GAR0072, Multisciences). The protein bands were visualized by High‐sig ECL Western Blotting Substrate (Tanon). Images were collected using the Tanon‐5200 Chemiluminescent Imaging System (Tanon). The expression of β‐actin was detected as a loading control.
All primary antibodies were commercial products. Anti‐TAB182, anti‐CDC2 (sc‐137035), anti‐phospho‐CDC2 (sc‐136014) were purchased from Santa Cruz. Anti‐FHL2 was purchased from Proteintech. Anti‐CHK2 (ab207446), anti‐phospho‐CHK2 (ab32148) were purchased from Abcam; anti‐CDC25C (YM0142), anti‐phospho‐CDC25C (YP0058) were purchased from ImmunoWay. Anti‐β‐actin (ab008) was purchased from Multisciences.
All primary antibodies were commercial products. Anti‐TAB182, anti‐CDC2 (sc‐137035), anti‐phospho‐CDC2 (sc‐136014) were purchased from Santa Cruz. Anti‐FHL2 was purchased from Proteintech. Anti‐CHK2 (ab207446), anti‐phospho‐CHK2 (ab32148) were purchased from Abcam; anti‐CDC25C (YM0142), anti‐phospho‐CDC25C (YP0058) were purchased from ImmunoWay. Anti‐β‐actin (ab008) was purchased from Multisciences.