Full-length FAK (2 ng/μl, #PV3832, Life Technologies, USA) and active PKM2 (6 ng/μl, #6372, BioVision, CA, USA) in the binding buffer (0.05% BSA, 5 mM DTT, 0.05% Triton X100 and 1x PBS, pH 6.5) were mixed and incubated at 4°C for 1 hr. Then, anti-PKM2 antibody (#4053, Cell Signaling Biotechnology, MA, USA) or anti-FAK4.47 was added to the mixture and incubated at 4°C for 2 h. In addition, AG-agarose plus was added, and the mixture was incubated at 4°C overnight.
Immunoprecipitates or cell lysates in NP-40 lysis buffer containing protease and phosphoratease inhibitor cocktails (Roche, USA) were loaded, and gels run as previously described. 30 , 37 (link) Band intensities were quantified using Image-J software (NIH) and normalized to the levels of total FAK, GAPDH or β-actin intensity.
Immunoprecipitates or cell lysates in NP-40 lysis buffer containing protease and phosphoratease inhibitor cocktails (Roche, USA) were loaded, and gels run as previously described. 30 , 37 (link) Band intensities were quantified using Image-J software (NIH) and normalized to the levels of total FAK, GAPDH or β-actin intensity.