Rabbit monoclonal anti human stim1 antibody

Western blotting was performed to confirm the suppression of STIM1 protein after dsiSTIM1 transfection. Protein samples (30 µg) were analyzed by SDS-PAGE on 12% gel. Following electro-blotting to polyvinylidene difluoride membranes, the membranes were blocked in 5% non-fat dry milk or 3% bovine serum albumin (BSA) in 0.1% Tris-buffered saline with Tween-20 (TBST) for 1 h at room temperature. Membranes were rinsed in 1X TBST three times and incubated in primary antibody solutions overnight at 4 °C with gentle rocking. The primary antibodies included rabbit monoclonal anti-human STIM1 antibody (Cell Signaling Technology, Danvers, MA, USA) at 1:500 dilution in 5% non-fat dry milk in 0.1% TBST and rabbit monoclonal anti-human β-actin antibody (Cell Signaling Technology, USA) at 1:2000 dilution in 3% BSA in 0.1% TBST. The membranes were washed the next day with 1X TBST three times and incubated in HRP-conjugated polyclonal anti-rabbit secondary antibody (Cell Signaling Technology, USA) at 1:500 dilution in 0.1% TBST including non-fat dry milk or BSA for 1 h at room temperature. After washing with TBST, the membranes were incubated in ECL substrate (Bio-Rad, USA) according to the manufacturer’s directions. After incubation, membranes were imaged using the VersaDoc imaging system (Bio-Rad, USA). Band intensity was measured using Image Lab software version 6.1 (Bio-Rad, USA).