Direct load color prestained protein marker m221

Western blot (WB) was performed as previously described.29 (link) Briefly, cells were harvested and lysed in RIPA buffer supplemented with protease and phosphatase inhibitor cocktails (1%, Sigma-Aldrich) on ice, followed by centrifugation and denaturation. The proteins were separated through SDS polyacrylamide gel electrophoresis and transferred to PVDF membranes. After being blocked in 5% BSA for 1 hour at room temperature, the membranes were incubated with primary antibodies at 4°C overnight and secondary antibodies at room temperature for 1 hour. Then electrochemiluminescence determination (PerkinElmer) was employed to detect the protein levels. Primary antibodies used were as followed: LRIG2 (1:500, Abcam, ab121472), sLRIG2 (1:750, Abcam, ab157492), ADAM17 (1:750, PAB30220), FLAG (1:1000, Sigma, F1804), CD47 (1:1000, Abcam, ab175388), c-Jun (1:1000, CST, #9165), c-Jun (phospho S63) (1:1000, Abcam, ab32385), anti-GAPDH (1:20000, Abclonal, AC002, RRID:AB_2736879), β-Actin(1:10000, Abclonal, AC026), anti-Albumin (1:1000, Abclonal, A0353). And Direct-load Color Prestained Protein Marker (M221, GenStar) was used in all WB assays.