Mouse anti flag or anti gapdh antibodies

The cells were collected and lysed using ice-cold lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% SDS, 1 mM EDTA, 1% NP-40) containing 1 mM protein inhibitor and 1 mM PMSF, for 30 min on ice. The lysates were centrifuged at 10,000 x g at 4°C for 10 min and the supernatants were collected. Protein concentration was measured using the BCA protein assay (HyClone-Pierce, Rockford, IL, USA). Equal amounts of total protein of each treatment were separated using 12.5% SDS-PAGE according to Laemmli's method (19 (link)), and were then transferred onto PVDF membranes. Membranes were incubated with mouse anti-FLAG or anti-GAPDH antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Western blotting was developed using horseradish peroxidase-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology) and was detected by enhanced chemiluminescence (ECL) reagent (ECL-Plus/Kit; Amersham, Piscataway, NJ, USA).