FIG. 4.

Downregulation of CaMKKβ and LKB1 by RNA interference. HUVEC were incubated in the presence of synthetic RNA duplexes targeted to human CaMKKβ or human LKB1 (1 μg/30-mm dish, 72 h). Incubations with transfection reagent only (control) or with a control RNA duplex containing an unrelated sequence (control siRNA) were run in parallel. A. Protein expression was examined in anti-CaMKKβ immune complexes (left panel) or cell lysates (right panel) probed with either anti-CaMKKβ antibody or anti-LKB1 antibody, respectively. Representative Western blots out of three independent experiments for each treatment are shown. B. CaMKKβ or LKB1 activities were determined in anti-CaMKKβ or anti-LKB1 immune complexes isolated from 100 μg cell lysate. Results are plotted as AMPK activity stimulated by the immune complex and measured using the SAMS peptide (U/mg recombinant AMPK, where 1 unit equals 1 nmol ³²PO₄ incorporated into SAMS peptide per min). Means ± SEMs for three independent experiments are shown. , P < 0.05 versus control siRNA.