Fig. 2. Quercetin decreases the expression of OGT, O-GlcNAc, and O-GlcNAcylated AMPK. Representative western blot analysis and relative bar graph quantification of OGT (A) and O-GlcNAc (B) in HeLa and HaCaT cells after treatment with 50 µM quercetin for 24 hours. (C) Upper panel: western blots using anti-O-GlcNAc and AMPK antibodies, of O-GlcNAcylated proteins precipitated by agarose-bound succinylated WGA beads, from HaCaT and HeLa cells treated with or without 50 µM quercetin for 24 hours. The specificity of WGA binding was confirmed by adding GlcNAc. Lower panel: relative bar graph quantification of relative O-GlcNAc levels. (D) Upper panel: western blots, using anti-O-GlcNAc and AMPK antibodies, of AMPK immunoprecipitated from cell lysates of HaCaT and HeLa cells treated with or without 50 µM quercetin for 24 hours. Lower panel: relative bar graph quantification of relative O-GlcNAc levels. Densitometry of immunoprecipitated AMPK to O-GlcNAc was normalised to IgG. OGT, O-linked N-acetylglucosamine transferase; O-GlcNAc, O-linked N-acetylglucosamine; AMPK, AMP-activated protein kinase; WGA, wheat germ agglutinin; SEM, standard error of mean; CTL, control; Quer, quercetin; IB, immunoblot. Each experiment was performed three times. Data represent the mean±SEM of three independent experiments. ^*P<0.05, ^**P<0.005, ^***P<0.001.