Rabbit anti orai1

Lysates were prepared using RIPA lysis buffer (Sigma) supplemented with protease inhibitors (Thermofisher Scientific). 30 and 50 μg of protein lysate was used for experiments with HEK293 and MCF7 cells respectively. Protein was loaded into a 4–12% Bis-Tris gel (Life Technologies) and then transferred onto a PVDF membrane (Millipore). After blocking for 1 hr. at room temperature with Olympus Blocking Buffer (LI-COR), membranes were incubated for 2 hours at room temperature with primary antibody diluted in Olympus Blocking Buffer and 0.1% Tween. The dilutions of primary antibodies were: rabbit-anti-ORAI1 (Sigma) 1:1000, mouse-anti-HSP70 (Santa Cruz) 1:4000, mouse-anti-GAPDH (Millipore) 1:3000, and mouse-anti-α-tubulin (CST) 1:2000. Following four washes with 0.1% TBST for five min. each, membranes were placed in secondary antibody diluted in Olympus Blocking Buffer and 0.1% Tween for 1 hr at room temperature. The secondaries were 1:10,000 680RD conjugated anti-mouse (LI-COR) and 1:5000 800CW conjugated anti-rabbit (LI-COR). After four further washes with 0.1% TBST for five min. each, and images of blots were taken using Odyssey Clx imaging system. Densitometry was quantified using ImageStudio Lite.