Figure 4.

Selective Orai3 plasma membrane accumulation is evoked by exogenous arachidonic acid (AA) and depends on microsomal glutathione S-transferase 2 (MGST2). A, Mean immunofluorescence data for α-Orai3-labeled Orai3 in cells treated with sorafenib (1 μmol/L), vehicle or cytosolic group IV phospholipase A2α (cPLA2α) inhibitor (1 μmol/L) before stimulation with vascular endothelial growth factor (VEGF; 30 ng/mL) for 10 minutes (n=4/N=15 each). B, Representative images and mean data (n=4/N=22 each) of cells treated with sorafenib before stimulation with vehicle or AA (40 μmol/L) for 10 minutes. Cells were labeled with anti-Orai3 (green) and anti-CD31 (red) antibodies. Scale bar, 10 μm. Arrows point to cell perimeter as indicated by signal for CD31. C, Mean data for cells overexpressing Orai3-[HA] and treated as in (B; n=3/N=15 each). D, Representative images and mean data for mCherry-Orai1 surface localization in cells treated as in (B; vehicle control, n=7/N=29; AA, n=3/N=16 each). Scale bar, 10 μm. E, Representative immunoblot and mean data from 3 experiments for cell surface Orai3 from cells treated as in (B) before biotinylation. The arrow points to Orai3 labeled by anti-Orai3 antibody (α-Orai3). The protein band above it, labeled nonspecifically by α-Orai3, has unknown identity. Where indicated (+), sorafenib and AA were used at 1 μmol/L and 40 μmol/L, respectively. AA was applied for 5 minutes. F, Representative immunoblot and mean data from 3 experiments for cells treated with VEGF (30 ng/mL) for 5 minutes before biotinylation. The arrow points to Orai3 labeled by anti-Orai3 antibody (α-Orai3). The protein band above it, labeled nonspecifically by α-Orai3, has unknown identity. Where indicated (+), cells were transfected with control scrambled (scr.) siRNA or MGST2 siRNA_1. All data are from HUVECs. Data are represented as mean±SEM. STIM1 indicates stromal interaction molecule 1. *P<0.05; not significant (ns) P>0.05.