Transcript expression and protein content of key players involved in skeletal muscle calcium homeostasis. (A) Expression levels of transcripts encoding ECC proteins measured by qPCR. Values are plotted as mean (±SEM) fold change in ryr3−/− vs. WT levels (which were set as 1). ACTN2 was used as housekeeping gene. The mean expression level of duplicate determinations obtained from pooled EOM from four to six mice is shown. Transcript levels of RYR1 and CASQ1 were reduced by ∼50%. *, P < 0.05, Student’s t test. (B) Left: Representative immunopositive bands obtained from Western blots of EOM total homogenates probed with the indicated antibodies. Right: mean (±SEM) intensity values of the immunopositive bands normalized for desmin (except for MyHC13 that was normalized for total MyHC content). The mean intensity values were obtained from EOM muscles from four to eight mice. The intensity values of obtained from WT mice were set to 100%. *, P < 0.05, Student’s t test. (C) The following commercial antibodies were used: rabbit anti-RyR1 (D4E1; 8153S; Cell Signaling), goat anti-Cav1.1 (sc-8160; Santa Cruz), rabbit anti-Cav1.2 (sc-25686; Santa Cruz), rabbit anti-calsequestrin-1 (CASQ1; C-0743; Sigma) and calsequestrin-2 (CASQ2; ab-3516; Abcam), goat anti-SERCA1 (sc-8093; Santa Cruz), goat anti-SERCA2 (sc-8095; Santa Cruz), mouse anti-MyHC (05–716; Millipore), mouse anti-MyHC13 (4A6; DSHB Iowa), and rabbit anti-parvalbumin (PV25; Swant). The rabbit anti-JP-45 polyclonal antibodies have been characterized previously (Zorzato et al., 2000). MW, molecular weight.
