Figure 1.

Ca_vβ₂ downregulation enhances PE-induced hypertrophy of NRCs. (A) RT-PCR analysis of the expression of the four Ca_vβ isoforms in NRCs and adult brain. The lower panel shows the expression of the different Ca_vβ₂ splice variants in NRCs. (B) Scheme of the domain organization of Ca_vβ₂, showing the regions where the designed shRNAs bind: NT (N-terminal), SH3 (Src-3 homology domain), GK (guanylate kinase domain), CT (C-terminal). (C) Representative western blot analysis of Ca_vβ₂ expression in homogenates from NRCs transduced with the different shRNAs for Ca_vβ₂-silencing and the scrambled shRNA (shRNAsc) used as negative control. Cells were transduced with each shRNA at an adenoviral MOI of 5 or 20. Per lane, 50 μg of protein were loaded. An anti-GAPDH western blot was used as loading control. (D) Bar plot of Ca_vβ₂ expression normalized vs. GAPDH in homogenates from NRCs transduced with the different shRNAs for Ca_vβ₂-silencing and the scrambled shRNA (shRNAsc.) at an adenoviral MOI of 5 or 20. Mean ± SEM from 3 replicated experiments; ^##p < 0.05 vs. shRNAsc treated at the same MOI (two-way ANOVA with Holm–Sidak's method). (E) Representative fluorescent images of control and Ca_vβ₂-downregulated NRCs (shRNA892) treated with vehicle or 50 μM phenylephrine (PE). Red, α-actinin; Blue, nuclear staining. Scale bar represents 15 μm. (F) Bar plot of the mean values of the cell area of control and Ca_vβ₂-downregulated NRCs (shRNA892) after treatments with vehicle or PE. Mean ± SEM; 150–200 cells per group from 20 randomly chosen fields from 4 replicated experiments were measured; ^§§p < 0.01 vs. vehicle-treated cells in the same group; ^§p < 0.05 vs. vehicle-treated cells in the same group; ^##p < 0.05 vs. control treated similarly (two-way ANOVA with Holm–Sidak's method).