ER vesicles from HEK293 cells expressing WT or mutant RyR1 were prepared by homogenizing cell pellets on ice using a Teflon-glass homogenizer with two volumes of solution containing 20 mmol/L (mM) Tris-maleate (pH 7.4), 1 mM EDTA, 1 mM DL-Dithiothreitol (DTT) and protease inhibitors (Roche). Homogenate was then centrifuged at 4,000 xg for 15 min at 4 °C and the resulting supernatant was centrifuged at 40,000 xg for 30 min at 4 °C. The final pellet, containing the ER fractions, was resuspended and aliquoted in 250 mM sucrose, 10 mM MOPS (pH 7.4), 1 mM EDTA, 1 mM DTT and protease inhibitors. Samples were frozen in liquid nitrogen and stored at −80 °C.
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