RNAi confirms the role of sAC in glucose-induced cAMP production. (A) Western blot, along with quantitation, using sAC-specific mAb R21 (top) or anti–β-actin mAb (middle) of INS-1E cells transfected with no RNA (mock; white bar), sAC-specific RNAi oligonucleotide (sAC-siRNA; black bar), or nonspecific oligonucleotide (Neg C; gray bar). sAC bands from two independent transfections were quantified by densitometry and normalized to β-actin (bottom). (B) Total cellular cAMP in untransfected INS-1E cells (Un) or INS-1E cells transfected with no RNA (mock), sAC-specific RNAi (sAC siRNA), or nonspecific oligonucleotide (neg C) after incubation for 15 min in Krebs Ringer buffer with low (2.5 mM; white bars) or high (16 mM; black bars) glucose. Values represent means ± SEM (n = 6) of total cAMP content per well, where each well contained equivalent number of cells upon RNAi transfection. High glucose stimulated cAMP production 1.78-fold in controls (untransfected, mock-transfected, and negative control–transfected cells) and 1.34-fold in sAC-siRNA-transfected INS-1E cells. ANOVA statistical analysis was performed with the Newman-Keuls post-hoc test. ns, not significant; **, P < 0.01; ***, P < 0.001.