Immunoprecipitation and Western blot

For immunoprecipitation studies, CHO cells were lysed on ice by scraping in lysis buffer containing 20 mM Hepes (pH 7.4), 100 mM NaCl, 1 mM MgCl₂, 1 mM EDTA, 1% Triton X-100, and protease inhibitors, followed by constant agitation for 30 min at 4°C. Protein extracts were centrifuged at 10,000g for 10 min at 4°C. For YFP-HA-Orai1 pulldown, 1 mg of lysate protein from YFP-HA-Orai1–transfected cells was immunoprecipitated using anti-GFP microbeads, as described previously. Extracts from cells transfected with pEGFP-C1 plasmid were used as a negative control when necessary. For endogenous Orai1 immunoprecipitation, 1 mg of lysate protein was precleared by incubation with 1 μg of normal rabbit IgG coupled to 50 μl of protein A+G agarose beads (Thermo Fisher Scientific) for 1 hour with constant agitation, followed by centrifugation at 5000g for 1 min. Orai1 was immunoprecipitated from the precleared lysate with 2 μg of rabbit polyclonal anti-Orai1 antibody (Abcam) coupled to 50 μl of protein A+G agarose beads by gentle rotation at 4°C overnight. In some experiments, lysates from GFP-CCT2–expressing cells were used. CCT2 and CCT5 were immunoprecipitated using CCT2 (Sigma-Aldrich) and anti-CCT5 (GeneTex) antibodies, respectively.

For Western blot analysis, protein extracts were obtained from cells lysed in cold lysis buffer containing 25 mM tris-HCl (pH 7.6), 150 mM NaCl, 1% IGEPAL CA-630, and protease inhibitors for 30 min, followed by centrifugation at 10,000 rpm for 10 min at 4°C. Protein concentrations were determined using a Bio-Rad protein assay. Equal amounts of proteins were loaded on 4 to 12% SDS-PAGE. Electrophoresed samples were transferred to a PVDF membrane and blocked with 5% milk in TBST (tris-buffered saline with Tween 20), and the membrane was incubated overnight at 4°C with primary antibody with constant shaking. After washing, horseradish peroxidase–conjugated secondary antibody was applied. Bound proteins to the secondary antibody were visualized using ECL (Amersham Biosciences). Quantitative Western blot analysis was performed using the secondary IRDye 800CW goat anti-mouse IgG and IRDye 680RD goat anti-rabbit IgG antibodies, and blots were scanned on a LI-COR Odyssey platform, followed by analysis using LI-COR Image Studio Lite v. 4.0.

For analysis of the subcellular fractions, an equal sample volume (30 μl) was mixed with an equal volume of 2× SDS loading buffer loaded onto 4 to 12% SDS polyacrylamide gels. Proteins were transferred onto PVDF membranes at 20 V for 1 hour, probed with primary antibodies, and visualized by ECL using horseradish peroxidase–conjugated secondary antibodies. For proteomics studies, lysates from fractions were resolved by electrophoresis on 4 to 12% SDS polyacrylamide gels and stained with Coomassie Brilliant Blue R-250.