Protease inhibitor cocktail

Plasmids (1 mg/ml) expressing HA-Cpgp40 (pcDNA3.1-HA-Cpgp40) and ENO1 (pcDNA3.1-ENO1) were mixed at the ratio of 1:1 and transfected into HEK293 cells in three-well plates using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions. As controls, cells were also transfected with plasmids pcDNA3.1-ENO1 or pcDNA3.1-HA-Cpgp40 under the same conditions. At 48 h post-transfection, cells were lysed in weak RIPA lysis buffer (Solarbio, China) containing 1 mM phenylmethylsulfonyl fluoride (Solarbio, China) and protease inhibitor cocktail (Solarbio, China). The cell lysates were incubated with mouse anti-ENO1 antibody or anti-HA antibody (Abcam, UK) at 4 °C overnight, followed by antibody capture by Protein G agarose beads (Santa Cruz Biotechnology, CA) at 4 °C for 3 h. The agarose beads were then extensively washed with weak RIPA lysis buffer containing 1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail and PBS containing 1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail. Proteins bound to agarose beads were solubilized in 1× SDS-loading buffer, boiled at 100 °C for 10 min, and examined by immunoblotting analysis.

Cells were washed twice in ice-cold PBS and lysed in lysis buffer (40 mM Tris-HCl, pH8, 5 mM MgCl₂, 40 mM Na4P207, 1% Triton X-100, 10 mM EDTA, 50 mM NaF, 100 µM Na3VO4, 1/25 protease inhibitor cocktail (ChemCruz)). Equal protein amounts of each lysate were separated on 7.5% SDS/PAGE gels before immunoblotting, as previously described [12 (link)], with the indicated antibodies.