Fig. 2: In vitro phosphorylation of RUNX2 is AMPK specific.
a p-AMPK substrate motif-specific antibody showing phosphorylation of GST-RUNX2-WT but not in lanes of serine-specific mutant of RUNX2-S118A, and GST control and b confirmed by [γ-32P] autoradiogram. Phosphorylation bands were detected in N-terminal protein (1–140) lane only. Lane RUNX2–R115A shows decreased intensity bands and explains the significance of highly conserved charged amino acids in efficient binding of AMPK. c Immunoprecipitation analysis showing the physical interaction of endogenous RUNX2 with AMPK. d Increased immunoprecipitation was seen by active AMPK with RUNX2 in either metformin or AICAR-treated C2C12 cells compared with compound C treatments. e Colocalization studies by immunofluorescence staining confirms the physical interaction, RUNX2 (Alexa Fluor 488) AMPK (Alexa Fluor 546) and Nuclei (DAPI) and f the quantification was performed using Image-J software on three independent experiments and fields. g Electrophoretic mobility shift assay was performed using nuclear lysates from RUNX2 full length overexpressed followed by metformin (5 mM) and compound C (20 µM) treatment in HEK 293T cells. The subsequent nuclear extracts overexpressed (RUNX2-WT, S118A and S118D) were incubated with osteocalcin promoter-specific probe. Mean  ± S.E.M.; n = 3,**p < 0.01 versus control
