Mouse anti ryr1 antibody

HeLa cells transfected wit RyR3-or control empty vector pcDNA were grown for 24-36 h, washed with ice-cold PBS and extracted with RIPA buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1% TX-100; 0.1 % SDS and 0.5% deoxycholate) for 30 min. After centrifugation at 13,000 rpm for 5 min, the supernatant was diluted in Laemmli sample buffer and 50 µg protein samples were separated by PAGE. Membrane was first probed overnight with a mouse anti-RyR1 antibody (1:5000; ThermoFisher) followed by a horseradish peroxidase-labelled secondary antibody (1:1000; Bio-Rad) and incubated for 1h. To control for protein loading, membrane was probed with a mouse anti-tubulin antibody (1:5000; Sigma). Sample proteins were quantified by the Bradford assay.

Stable HeLa clone expressing erGAP3 (see below) were seeded on 12 mm coverslips and transiently transfected with RyR3 cDNA. Cells were fixed for 24-36h with 4% PFA in phosphate buffered saline (PBS) for 20 min; 10% normal goat serum was added for blocking nonspecific binding sites. Expression of RyR3 was detected by incubating the mouse anti-RyR1 antibody (1:200; ThermoFisher) diluted in PBS and containing 10% goat serum, overnight at 4ºC. After washing with PBS, the secondary Alexa Fluor 568conjugated antibody (1:200; Molecular Probes) was added and incubated for 60 min at 22ºC. Cultures were washed with PBS three times and mounted in Vectashield (Vector).
GAP was detected as green fluorescence (excited at 470/40 nm and filtered at 540/50 nm) in a Zeiss Axioplan Z microscope equipped with a 63x/1.2w Korr objective and an AxioCam MR camera. The red fluorescence was excited at 560/40 and filtered at 605/50 nm). The Zeiss ApoTome R system was used for optical sectioning and images were analysed with AxioVision and Image J software.