Immunoprecipitation and Immunoblotting of Cellular Proteins

Liver lysates upon hepatectomy surgery and HEK293 cells with indicated treatments were lysed using a modified Myc lysis buffer (MLB) (20 mM Tris-Cl, 200 mM NaCl, 10 mM NaF, 1 mM NaV₂O₄, 1% NP-40, 20 mM β-glycerophosphate, and protease inhibitor (pH 7.5)) [77 (link)]. Cell lysates were then subjected to immunoprecipitation using antibodies of anti-Flag (Sigma-Aldrich, F3165-5MG, 1:200 dilution), anti-Myc (2276S, Cell Signaling Technology, 1:200 dilution), or anti-HA (3724S, Cell Signaling Technology, 1:200 dilution) for transfected proteins, or using anti-PPM1A (3549, Cell Signaling Technology, 1:100 dilution) antibody for endogenous proteins. After 3 to 4 washes with MLB, adsorbed proteins were resolved by SDS-PAGE (Bio-Rad) and immunoblotting with the indicated antibodies. Cell lysates were also analyzed using SDS-PAGE and immunoblotting to control the protein abundance.