Complete mini protease cocktail

Caco-2 cells from two 10-cm cell culture plates were washed with phosphate-buffered saline (PBS) and harvested by scraping. Next, the cells were lysed in 1.5 ml lysis buffer (25 mM HEPES, pH 8.0, 100 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 20 mM β-glycerophosphate, 20 mM para-nitrophenyl phosphate, 100 μM ortovanadate, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 1X cOmplete mini protease inhibitor cocktail [Roche, Switzerland]), homogenized by passing the cells through a 23G needle 10 times and rested 45 min on ice. For immunoprecipitation, cell lysates were incubated with rabbit anti-lamin A (Abcam, UK) or mouse anti-K18 (L2A1; Bishr Omary, Rutgers University) antibody (not added to control samples) under rotation at 4°C overnight. Protein-A/G magnetic beads (30 μl; Thermo Fisher Scientific, Waltham, MA) were added to each sample and the samples were incubated under rotation at 4°C for 20 min. The samples were washed three times in TEG buffer (20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 10% glycerol, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol and 1X cOmplete mini protease cocktail; Roche, Switzerland), dissolved in 3X laemmli sample buffer, and analyzed by SDS–PAGE and Western blot.