Western Blot Analysis and IP Protocol

Cells were lysed using cell lysis buffer for Western blot analysis and IP (Beyotime, Shanghai, China, P0013) supplemented with 1% PMSF (Beyotime, Shanghai, China, ST505). BCA Protein Assay Kit (Beyotime, Shanghai, China, P0010) was used to measure protein concentration. 12% SDS-PAGE was used to resolve proteins. Proteins were electrophoretically transferred onto PVDF Western blotting membranes (Roche, Mannheim, Germany, 3010040001). Proteins in the membranes were analyzed by enhanced chemiluminescence (ECL) reagents (Yeasen, Shanghai, China, 36208ES76) as described previously [42 (link)].
Primary antibodies were anti-Flag (mouse) (Abmart, Shanghai, China, M20008H), anti-Tubulin (Rabbit) (Beyotime, Shanghai, China, AF0001), and anti-BmCyclin B (Rabbit), which was previously prepared in our laboratory. HRP-labeled goat anti-mouse (or anti-rabbit) IgG (H + L) was applied as secondary antibody. Protein markers were Blue Plus^TM protein marker (TransGen, Beijing, China, DM111-01) and EasySee^TM Western Marker (TransGene Biotech, Beijing, China, DM201-02).