Figure 3.

Gene knockdown does not alter acute D₂ signaling but decreases the maximum efficacy of AC sensitization response in HEK-AC5/D_2L cells. Biochemical validation of siRNA knockdown was carried out by qPCR (A,C) and western blotting (B,D). Cell fractions from mock or siRNA-treated cells were subjected to western blot analysis using target antibodies for (B) PPP2CB and (D) NAPA, as well as listed loading controls. Data were standardized to mock transfection (left) and representative immunoblots (right). Statistical analysis was performed (t-test), *** p < 0.001. The ability of the Gα_i/o-coupled D₂ receptor to inhibit AC remained intact following gene knockdown (E). Quinpirole (3 μM) maximally inhibited the AC response to acute stimulation with 3 μM forskolin under all siRNA conditions. siRNA knockdown of target genes reduced the maximal efficacy of the AC sensitization response without affecting the potency of quinpirole (F). Experiments (n = 3) were performed in duplicate with SEM shown.