Adenoviral knockdown of Ca_V1.2 reduces PKA-II activity. (A) Representative HCS microscopy images of mouse DRG neurons transduced with AAV-PHP.S-U6-shRNA:Scramble-CAG-GFP (Scr) or AAV-PHP.S-U6-shRNA:Cacna1c-CAG-GFP (Cac) to knock down Ca_V1.2. Neurons were transduced after overnight culture (1 div) and fixed 1 wk (8 div) or 2 wk (15 div) later. Cultures were immunolabeled for the neuronal marker UCHL1 and pRII to quantify PKA-II signaling activity. The expression of GFP indicated efficient transduction. Nuclei were stained with Hoechst 34580. Scale bar, 100 µm. (B and C) GFP expression levels in individual neurons (left) and mean numbers of GFP⁺ neurons after 8 div (n = 6, >6,000 neurons per condition, Student’s t test) as well as after 15 div (n = 6, total of >1,000 neurons per condition, Student’s t test). (D and E) Single-cell data of all analyzed neurons (left) and mean intensities in GFP⁻ and GFP⁺ neurons (right) obtained using a Ca_V1.2-specific antibody (clone N263/31) at 8 div (D; n = 4, >4,000 neurons) and 15 div (E, n = 3, >1200 neurons) indicating down-regulation of Ca_V1.2 in GFP+ neurons. The primary antibody was omitted in respective controls (w/o AB). (F and G) Effect of AAV-mediated Ca_V1.2 knockdown on pRII intensity levels induced by 3-min stimulation with KCl at 8 div (F; n = 6, >6,000 neurons, two-way ANOVA with Bonferroni’s test) and 15 div (G, n = 6, >1,000 neurons per condition). (H) Cell density plots showing single-cell data of pRII intensities versus GFP expression after 15 div as shown in G. Values in B–G are means ± SEM; *, P < 0.05; ***, P < 0.001.