Anti phosphor ser thr antibody

For immunoblotting assay, cell lysis buffer (50 mM Tris-HCl pH7.4, 250 mM NaCl, 0.5% Triton X-100, 10% Glycerol, 1 mM DTT) containing a protease inhibitor cocktail (Roche) was used to lyse cells. For immunoprecipitation (IP) assay, E1A lysis buffer (250 mM NaCl, 50 mM HEPES pH 7.5, 0.1% NP40, 5 mM EDTA) containing a protease inhibitor cocktail was used to lyse cells. After sonication for 10 s for three times, cell debris were removed by spin down at 13,000 rpm for 15 min. Clarified cell lysates were then incubated overnight with magnetic beads pre-incubated with indicated antibody. The beads were washed with PBST for 6 times before eluting with SDS sample buffer and subjected to immunoblotting analysis. All the blots were quantified by ImageQuant TL software. The main antibodies included in the study were listed below: anti-p-AMPK (T172) (1:1000), AMPK (1:1000), anti-p-ACC (S79) (1:1000), anti-ACC (1:1000), anti-PDHK1 (1:1000) from Cell Signaling Technology; anti-PDHK2 (1:1000), anti-PDHK3 (1:1000), anti-Flag (1:3000) from Sigma; anti-HA (1:5000) from Covance; anti-GRP78 (1:5000) from BD Transduction Laboratories; anti-phosphor-Ser/Thr antibody (1:1000), anti-p-PDHA(S293) (1:1000) from Abcam; anti-PDHA (1:1000) from Santa Cruz. anti-p-PDHA(S295), anti-p-PDHA(S314) were generated by Proteintech Inc.