Detecting p63 Isoform Complexes by BN-PAGE

H1299 cells were transfected using the Qiagen Effectene kit according to the manufacturer’s manual in a 12-well plate or H1299 cells stably expressing p63 isoforms were used. Cells were harvested and lysed in NP lysis buffer (20 mM Tris, 150 mM NaCl, 2 mM MgCl₂, 20 mM CHAPS, 1 mM DTT, complete protease inhibitor cocktail (Roche), PhosSTOP (Roche) pH 7.4 or pH 8.0). Benzonase (Merck) was added and samples were lysed on ice for 1 h. BN-PAGE (3–12%, Thermo Fisher Scientific) was performed and blotted as previously described^{17 (link),35 (link)}. Protein levels loaded on BN-PAGE were adjusted to equal p63 amounts by prior western blot analysis. SDS-PAGE to detect phosphoshifts were not input adjusted.