Cell Lysis and Immunoprecipitation for Protein Analysis

The experimental cells were lysed 24 to 48 h after transfection with the expression plasmids using 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 1% NP-40 containing cocktail inhibitors (Roche, USA). For immunoprecipitation, the lysates were incubated overnight with the anti-FLAG M2 affinity gel (Sigma, USA). Immunoblotting was carried out as described elsewhere (46 (link)). Briefly, the cells were collected and lysed by adding radioimmunoprecipitation assay (RIPA) lysis buffer together with protease/phosphatase inhibitor cocktail on an ice bath for 30 min and by tapping tubes every 10 min. The protein concentration was quantified by the Coomassie Plus protein assay reagent (Thermo Scientific, Rockford, IL, USA). The quantification of immunoblotting band intensity was performed with the ChemiDoc XRS⁺ Molecular Imager software (Bio-Rad, Philadelphia, PA, USA). The samples were separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. After blocking in Tris-buffered saline (TBS) containing 0.1% Tween 20 and 5% skim milk, the blots were probed with relative antibodies.