Anti stim1

Freshly excised mouse salivary glands were minced and lysed in RIPA buffer containing protease inhibitors. The protein lysates were mixed with tris–glycine sodium dodecyl sulfate sample buffer (Invitrogen) and loaded onto gels for Western blot analysis. Rabbit monoclonal anti-STIM1(1:1000 dilution, Cell Signaling Technologies, Danvers, MA), anti-Orail1 antibody (1:1000 dilution, custom generated^{23 (link)}), and mouse polyclonal anti–β-actin antibody (1:5000 dilution, Abcam) were used for immunoblotting. After incubation with secondary antibodies, signals were detected by chemiluminescence using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) and ChemiDoc imaging system (Bio-Rad).

Cells were harvested and the proteins were extracted with ice-cold RIPA lysis buffer (Beyotime, P0013D). The protein concentration in the lysates was determined using a BCA protein assay kit (Thermo Fisher Scientific, 23225). Equivalent amounts of protein (25–40 μg) were separated by 10% SDS-PAGE gels and transferred onto a nitrocellulose filter membrane (NC) membrane (Pall, 66485) and blocked with 5% non-fat milk in Tris-buffered saline with Tween 20 (TBST). Primary antibodies were incubated at 4 °C overnight, including anti-FLAG (Abmart, M20008, 1:5000), anti-Myc (Affinity, T0052 1:5000), anti-STIM1 (Cell Signaling Technology, 5668, 1:3000), anti-ORAI1 (Santa Cruz, sc-377281, 1:500), anti-TRPC1 (Proteintech, 19482, 1:1000), anti-E-cadherin (Cell Signaling Technology, 3195 S, 1:1000), anti-Vimentin (Cell Signaling Technology, 5741 S, 1:1000), anti-GAPDH (Proteintech, 60004-1-1 g, 1:10000), anti-NFATc1 (Santa Cruz, sc7294, 1:500), and anti-NFATc2 (Santa Cruz, sc7296, 1:500).

Protein samples were separated using SDS-polyacrylamide gel and transferred to nitrocellulose membranes. Blots were incubated with 1:1000 anti-STIM1 (Cell Signaling, #4916, Danvers, MA, USA), 1:1000 anticalcineurin (Cell Signaling, #2641, Danvers, MA, USA), 1:500 anti-Orai1 (Santa Cruz, sc-68895, Dallas, TX, USA), 1:1000 anti-NFATc3 (Santa Cruz, sc-8321, Dallas, TX, USA), and 1:500 antiphosphorylated NFATc3 (Santa Cruz, sc-365785, Dallas, TX, USA). The membranes were then incubated with horseradish peroxidase-linked secondary antibody. The immunoblots were identified with SuperSignal West Picochemiluminescent substrate (Thermo Fisher Scientific, Waltham, MA, USA). Band intensity was calculated using ImageJ (National Institutes of Health), and intensity data from cytoplasmic, membranous, and nuclear proteins of interest were normalized to 1:1000 α-tubulin (Santa Cruz, sc-23948, Dallas, TX, USA), 1:1000 pan-cadherin (Sigma-Aldrich, C1821, St. Louis, MO, USA), and 1:500 lamin B (Santa Cruz, sc-6216, Dallas, TX, USA), respectively. Antidesmin (1:500, Upstate, Cat. #04-585, Lake Placid, NY, USA) was used for immunoblotting atrial tissue proteins.

Isolated CD8⁺ T cells were incubated in the presence of 2 mM of Ca²⁺, fixed with paraformaldehyde (Sigma-Aldrich), permeabilized using Triton X-100 (Sigma-Aldrich) and stained with anti-PMCA (Abcam), anti-TMEM20 (SantaCruz Biotechnology) or anti-STIM1 (Cell Signaling Technology) antibody. Then the samples were stained with anti-rabbit IgG-Alex Fluor^® 488, anti-rabbit-IgG-Alex Fluor^® 546 or IgG-Alex Fluor^® 647 (Life Technologies, Gaithersburg, MD) for PMCA, STIM1 or TMEM20, respectively. The images were obtained using LSM 510 META Laser Scanning Microscopes (Carl Zeiss).

Protein extract was isolated from HL-1 cells using lysis buffer: 1 mg of protein was mixed with 1:1000 anti-STIM1 (Cell Signaling, #4916, Danvers, MA, USA) at 4 °C for 1 h and then incubated with Pierce™ protein A/G magnetic beads (Thermo Fisher Scientific, St. Peters, MO, USA) overnight at 4 °C. The immunoprecipitation matrix was washed twice with PBS containing 1% NP-40. The matrix-bound protein was eluted in sample buffer and then separated by 10% SDS-PAGE electrophoresis. The magnetic beads were saturated to capture all STIM1 proteins in the cell lysate. The immunoblot was done with 1:1000 anti-O-GlcNAc (MA1-076, Thermo Fisher Scientific, St Peters, MO, USA) and 1:1000 anti-STIM1 incubation overnight at 4 °C, and signals were detected by secondary antibodies with chemiluminescent visualization.