Anti orai2

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.

hMSCs were seeded onto coverslips in 4-well plates, cultured for a day and treated with LPS or poly(I:C). Subsequently, the cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde in PBS for 15 min and permeabilized with cold methanol for 5 min. Then, the samples were blocked with 3% bovine serum albumin for 1 h, incubated with rabbit polyclonal anti-IP₃R3 (1:100; Abcam, Cambridge, UK), rabbit polyclonal anti-Orai1 (1:100; Abcam) or rabbit polyclonal anti-Orai2 (1:100; Abcam) at 4 °C overnight. A subsequent incubation of the samples with Goat anti-rabbit IgG conjugated to Alexa 488 (1:100; Life Technologies, Carlsbad, CA) was performed for 30 min at 37 °C. Finally, the samples were mounted in the mounting medium Vectashield (Vector Laboratories, Burlingame, CA) and visualized with a Zeiss LSM 710 confocal microscope (Jena, Germany).

Cell lysates were prepared by sonication in lysis buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 5 mM MgCl₂, 1% Triton X-100 and a complete protease inhibitor mixture tablet (Roche Applied Science). Equal amounts of protein (30–50 μg) were subjected to 6 or 10% SDS-PAGE and blotted onto a PVDF membrane. The membranes were then incubated with anti-IP₃R3 (1:1000; Abcam) or anti-Orai2 (1:1000; Abcam) and anti-pancadherin (1:1000; Abcam) or anti-β-actin (1:10000; Sigma) for loading controls and signals were detected with ECL reagent.