Anti orai1

All plasmids used in this study were sequenced to confirm their identity. Crbn, Ampk, Slo, or HA-Ubiquitin constructs used in this study were described previously^{26 (link)}. Orai1 (MMM1013-202764440), Orai2 (MMM1013-202859855), and Orai3 (MMM1013-202842392) cDNA were purchased from Open Biosystems to construct expression vectors by a PCR-based strategy. Glutamine synthetase (GS) cDNA were synthesized from mRNA of the testis of mice and GS expression vectors were generated. The complete list of all primer sequences used in the study is provided as a Supplementary table. Anti-Flag (Sigma, F1804), anti-HA (Santa Cruz, sc-7392), anti-GST (Santa Cruz, sc-138), anti-Crbn (Sigma, HPA045910), anti-Orai1 (Santa Cruz, sc-68895), anti-Orai1 (Alomone labs, ACC-062), anti-Orai2 (Abcam, ab180146), anti-Ampkα (Cell signaling, #2532), anti-phospho-AMPKα (cell signaling, #2535), anti-Stim1 (Abcam, ab108994), anti-Cul4A (Abcam, ab92554), anti-DDB1 (Bethyl Laboratories, A300-462A), anti-phospho-raptor (Cell signaling, #2083), anti-raptor (Cell signaling, #2280), anti-phospho-S6K (Cell signaling, #9206), anti-S6K (Cell signaling, #9202), anti-Lamin B (Santa Cruz, sc-374015), anti-Erk2 (Santa Cruz, sc-154), anti-CD16/32 (BioLegend, #101302), anti-CD11b (BioLegend, #1010207), and anti-β-Actin (Santa Cruz, sc-1616) were purchased.

The total protein extracted from deendothelialized distal PA homogenate was subjected to 12% SDS-PAGE and then transferred to a polyvinylidene fluoride membrane (Millipore, MA). Following blockade with 5% BSA buffer for 2 h, membranes were probed with anti-STIM2 (1:200; Alomone Labs, Jerusalem, Israel), anti-Orai1 (1:200; Alomone Labs, Jerusalem, Israel), anti-Orai2 (1:200; Alomone Labs, Jerusalem, Israel), anti-TRPC1 (1:300; Abcam, MA), anti-TRPC4 (1:200; Alomone Labs, Jerusalem, Israel), anti-β-actin (1:500; Cell Signalling Technology, MA) primary antibodies overnight and horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000; Cell Signalling Technology, MA) for 1 h. The bands were visualized by enhanced chemiluminescence reagent (Thermo Fisher Scientific, CA). The optical density of each blot was quantified using Quantity One software (Bio-Rad, CA) and normalized to β-actin on the same membrane.

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was performed by loading 50 µg of total protein lysate (without nuclei) extracted from HEK-293 cells on 12% polyacrylamide gels. Following electrophoresis, separated proteins were transferred to methanol-activated polyvinylidene fluoride (PVDF) membranes with the Pierce G2 Fast Blotter (Thermo Fischer Scientific). Membranes were incubated in 5% nonfat milk in TBS-T buffer (15 mM Tris-HCl, 140 mM NaCl, 0.05% Tween20^®, pH 7.4) for 1 h at room temperature to block nonspecific protein binding. Subsequently, the membranes were incubated with primary antibodies overnight at 4 °C. The primary antibodies were anti-ORAI1 (Alomone, Jerusalemn, Israel: Acc-060), used at a 1/200 dilution, and anti-β-actin Sigma-Aldrich, St. Quentin Fallavier, France: A5441), used at a 1/5000 dilution. Following washes in TBS-T, the membranes were incubated for 1 h with horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit secondary antibodies diluted at 1/100,000 (Chemicon). Chemiluminescent detection of bound secondary antibodies was captured with Amersham Imager 600 (GE Healthcare Life Sciences) using the SuperSignal™ West Femto Maximum Sensitivity Substrate and the SuperSignal™ West Dura Maximum Sensitivity Substrate (Thermo Fischer Scientific, Illkirch, France).