Figure 5.

Muscle tissue expression of hCAPN3 at mRNA and protein levels in CAPN3 KO mice 20 weeks after gene delivery

(A and B) Relative expression levels of hCAPN3 in several skeletal and cardiac muscle tissue samples from the old (A) and young age groups of CAPN3 KO mice (B). A dose-dependent increase in CAPN3 mRNA transcripts was observed in the HD (6 × 10¹² vg, n = 12) treatment cohort compared with the LD (3 × 10¹² vg, n = 12) cohort for both age groups. GAPDH was used as the reference gene. Data are represented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, two-way ANOVA, Sidak’s multiple comparison test. (C and D) Representative western blot images from 4 mice show variable amounts of 94-kDa CAPN3 protein in quadriceps (C) and gastrocnemius (D) muscles from the HD cohort of the old age group. A calibration curve ranging from 15–2.5 μg (30%–5%) total protein from the corresponding UT WT C57BL/6 reference muscles was used for semiquantitative analysis of AAV-mediated CAPN3 levels in the corresponding treated CAPN3 KO mouse muscles. CAPN3 levels were detected using the CALP-12A2 antibody, which recognizes epitopes in muscle extracts from human and mouse. Assay sensitivity was optimized to reliably detect the CAPN3 protein in amounts as low as 2.5% of the WT control level. CAPN3 protein levels in the quadriceps (C) and gastrocnemius (D) are quantified as percent of the WT and represented individually as bar graphs, and the mean levels of all HD samples (n = 12) are given as mean ± SEM.