Skip to main content
. 2009 May 4;29(13):3582–3596. doi: 10.1128/MCB.01417-08

FIG. 6.

FIG. 6.

Inhibition of PKC-ζ blocks LKB1 S307 phosphorylation and LKB1 translocation from the nucleus to the cytoplasm. (A) Pharmacological inhibition of PKC-ζ with PKC-ζ-PS diminishes ONOO−-enhanced LKB1 phosphorylation in BAECs. Shown is Western blot analysis of AMPK and LKB1 phosphorylation in confluent BAECs that were preincubated with PKC-ζ-PS (PKC-PS) for 30 min followed by exposure to ONOO− (50 μM) for 15 min. The blot is a representative of six blots obtained from six individual experiments (n = 6). ♣, P < 0.05 versus control (Con). (B) Effect of PKC-ζ-PS on metformin-induced (Met, 1 mM, 1 h) LKB1 and AMPK phosphorylation in BAECs. The blot is representative of six blots obtained from six individual experiments (n = 6). ♣, P < 0.05 versus control; †, P < 0.05 versus Met. (C) Effect of PKC-ζ-specific siRNA on metformin-induced AMPK T172 phosphorylation in HUVECs (n = 5). ♣, P < 0.05 versus control; †, P < 0.05 versus metformin plus scrambled siRNA (S-siRNA). (D) Effect of PKC-λ/ι-specific siRNA on metformin-induced AMPK-T172 phosphorylation in HUVECs. The blot is representative of three blots obtained from three individual experiments (n = 3). ♣, P < 0.05 versus control. (E) Metformin-induced LKB1-S307 phosphorylation in BAECs overexpressing PKC-ζ-DN, PKC-ζ-CA, or GFP. The blot is representative of five blots from five individual experiments (n = 5). ♣, P < 0.05 versus control; †, P < 0.05 versus metformin. Black bars, P-AMPK; white bars, P-LKB1 S307; and striped bars, P-LKB1 S428. (F) LKB1 immunocytochemical staining in confluent HUVECs preincubated with or without PKC-ζ-PS (PKC-PS) and exposed to metformin (1 mM, 1 h). Nuclei were counterstained with DAPI. (G) Time course of phosphorylation of PKC-ζ, LKB1, and AMPK in HUVECs in response to metformin. HUVECs were treated with metformin (1 mM) at the times indicated. The phosphorylation of PKC-ζ, LKB1 S307, LKB1 S428, and AMPK was detected at the indicated times by Western blotting with respective antibodies. The blot is representative of three blots from three individual experiments.