Peroxidase labeled secondary antibody

Cells were lysed in 1% (v/v) Triton X-100 in 20 mM Tris-HCl (pH 8), 150 mM NaCl in the presence of Protease Inhibitor Cocktail Set III (Calbiochem) and 0.2 mg Na orthovanadate/ml. Postnuclear supernatants were resolved by SDS-PAGE and transferred to nitrocellulose. Alternatively, postnuclear supernatants were immunoprecipitated using RanBPM polyclonal antibody (Proteintech) and protein A Sepharose (GE Healthcare). Immunoblots were carried out using peroxidase-labeled secondary antibodies (GE Healthcare) and a chemiluminescence detection kit (Bio-rad Laboratories Inc., Milan, Italy). Immunoblots were scanned and quantitated using ImageJ software.

For analysis of protein contents, neutrophils, platelets, and mast cells were lysed in 100 mM NaCl, 30 mM Na-HEPES (pH 7.4), 20 mM NaF, 1 mM Na-EGTA, 1% Triton X-100, 1 mM benzamidine, freshly supplemented with 0.1 U/ml Aprotinin, 1:100 Mammalian Protease Inhibitor Cocktail, 1:100 Phosphatase Inhibitor Cocktail 2, 1 mM PMSF, and 1 mM Na₃VO₄ (all from Sigma-Aldrich). After removal of insoluble material, lysates were boiled in sample buffer. Whole cell lysates were run on SDS-PAGE and immunoblotted using antibodies against Syk (Clone N19; Santa Cruz) or β-actin (Clone AC-74; Sigma-Aldrich) followed by peroxidase-labeled secondary antibodies (GE Healthcare). The signal was then developed using the ECL system (GE Healthcare) and exposed to X-ray film.

HEK cells were treated with 0–500 μM diamide (Sigma-Aldrich) in 2 mM Ca Ringer’s for 20 minutes to induce the formation of disulfide bridges, then washed with cold PBS for 5 minutes. To quench unreacted thiols, cells were lysed in lysis buffer containing 50 mM N-ethylmaleimide (Sigma-Aldrich), 150 mM NaCl, 50 mM Tris, 1% Triton X-100, 0.1% SDS, and 1x protease inhibitor mixture (Roche) by incubating on ice for 15 min. Lysates were centrifuged at 4°C for 20 min and supernatants were collected. Isolated protein samples were heated to 65°C in Laemmli sample buffer (Bio-Rad) containing 0.1% mercaptoethanol and run on 4–20% SDS-polyacrylamide gradient gels (Bio-Rad). Protein was transferred onto nitrocellulose membrane and Orai1 was detected using 1:7500 purified monoclonal primary antibodies provided by Amgen (m266.1) [37 (link)] and 1:10000 peroxidase-labeled secondary antibody (GE Healthcare Life Sciences). GAPDH was blotted as a loading control. Monoclonal anti-GAPDH-Peroxidase (Sigma Alrdrich) antibody was the generous gift of the Volpert lab. Western blot images were analyzed using the image analysis software ImageJ. For CAD co-expression bots, WT-Orai1 and L276C-Orai1quantitations were quantitated at higher exposures due to their lower expressions compared to L273C-Orai1 channels under this condition. All conditions were quantitated at the highest exposure without saturation.