Figure 5. Assessment of LKB1 acetylation and phosphorylation in mouse adipose tissue. (A) Western blot of LKB1 post-translational modifications: acetyl-lysine, pS428, and pT336. LKB1 from mouse subcutaneous adipose tissues were immunoprecipitated, and LKB1 acetylation, pS428, and autophosphorylation site pT336 were assessed by western blotting. (B) LKB1 acetylation and pT366. The degree of acetylation of LKB1 is negatively correlated with pT336 (r=0.77, p<0.05), suggesting that LKB1 acetylation, which is regulated by Sirt1 activity, affects LKB1 activity. (C) Correlation between S428 and pT336. pS428 and pT336 were positively correlated (r= 0.824 p<0.05), suggesting that pS428 indicates activation (D). pS428 LKB1 is located in the cytosol. Two sets of samples were fractionated. LKB1 pSer428 was only observed in the cytosolic fraction, suggesting that LKB1 pSer428 is indicative of active (cytosolic) LKB1 content. Similar experiments were repeated three times.