Fig. 4. SOAR gates Orai1 but does not mediate clustering of STIM1 or co-clustering of STIM1-Orai1.

(a–b) CRAC current was measured in cells transfected with Orai1 and either SOAR (black), SOAR(LQ/AA) (red), STIM1 (blue) or STIM1(LQ/AA) (green). (b) is the mean±s.e.m. of 4 experiments. In (c), cells were co-transfected with mCherry-Orai1 and EGFP-SOAR(LQ/AA). In (d), cells were transfected with STIM1(LQ/AA) alone and were untreated (upper image) or treated with 25 µM CPA in Ca²⁺- free media for 10 min. (e) shows two examples of cells co-transfected with mCherry-Orai1 and EGFP-STIM1(LQ/AA) and treated with CPA for 10 min. Arrows in (e) point to co-localized Orai1-STIM1 in puncta. Each set of images represent at least 50 analyzed cells from 2 transfections. In (f, g), cells were transfected with Orai1 and SOAR or SOAR(LQ/AA) (f) or Orai1 and STIM1 or STIM1(LQ/AA) (g) and used to IP Orai1 with anti FLAG and probe for co-IP of SOAR or STIM1 with anti-GFP. Fig. S7b shows full blot of Orai1 and Fig. S7c shows full blot of EGFP-STIM1 and EGFP-SOAR. In addition, Fig. S7c is the control for the co-IP in panels (f, g). In (h), TRPC1 was expressed in control cells alone (condition 1, black) or together with STIM1 (condition 2, magenta), STIM1(LQ/AA) (condition 3, red), or in siSTIM1-treated cells alone (condition 4, green) or together with STIM1(LQ/AA) (condition 5, blue), and the carbachol-activated TRPC1-mediated Na⁺ current was measured. The columns show the mean±s.e.m. of the % current in 4 experiments.