Fig. 1. IP3R isotypes. (A) Left panel: RT-PCR results of IP3R-1, 2 and 3 RNAs (type 2 and 3 appear as one band) in cultured muscle cells: rat myotubes (Mr) and the mouse muscle cell line, C2C12 (C2); rat CER, used as a positive control for IP3R-1. RT+ and RT-, with and without reverse transcriptase. Products were separated by electrophoresis in 0.8% agarose. Right panel: same experimental conditions as shown in the left panel, but 3% agarose was employed and three bands of expected sizes were obtained. Base pairs (bp) are noted in A and B. (B) First-strand cDNA was transcribed from total RNA of rat myotubes using random hexamers as primers. PCR was performed with primers specific for each IP3R isoform 1, 2 and 3. The products were separated by electrophoresis in 3% agarose. RT+ and RT- as in A. (C) Western blot analysis of IP3R-1 and 3 in skeletal muscle in culture. Nuclei isolated from the cell line C2C12, and homogenates from rat skeletal muscle (Mr) in primary culture were analyzed for the presence of types 1 and 3 IP3Rs. Thirty µg of protein from nuclei and 30 µg (Mr1) or 60 µg (Mr2) of rat myotube homogenate were incubated with: anti-IP3R-1 antibody (top panel), where 2 µg of rat (CER) were used as a positive control; or anti-IP3R-3 antibody (bottom panel), using 10 µg of HeLa homogenate as positive control.
