Figure 7

ORAI1-CTTN co-localization and co-precipitation. (a) U2OS wild-type cells were transfected for the transient expression of ORAI1-GFP/mCherry-CTTN and monitored for both tags in Leibovitz’s L-15 medium supplemented with 10% FBS. Emission of fluorescence was recorded every 3 sec for 10 min, and images were split in both channels (GFP and mCherry). The GFP/mCherry ratio was calculated from single channels, and is shown on a pseudocolor scale (ratio values 0–2). A selected area was assessed for the variation of fluorescence intensity, shown in the line graph. The full time-lapse sequence is given in Supplementary Movie 8. (b) Endogenous ORAI1 was immunoprecipitated with an anti-ORAI1 antibody conjugated to agarose beads (lane 2). The level of immunoprecipitated ORAI1 was assessed with an anti-ORAI1 antibody (bottom panel), and the level of co-immunoprecipitated CTTN was evaluated with an anti-CTTN antibody (top panel). As a negative control, lysates from the same experimental conditions were incubated with preimmune serum and agarose beads (lane 1). Blots are representative of 3 independent experiments with 3 different cultures. (c) U2OS cells transiently expressing GFP (lane 1) or ORAI1-GFP (lane 2) were lysed to pull-down GFP-tagged ORAI1 with GFP-Trap beads. The level of endogenous CTTN bound to ORAI1-GFP was evaluated by immunoblot using an anti-CTTN antibody (top panel), and the level of pulled-down GFP or ORAI1-GFP was evaluated with an anti-GFP antibody (bottom panel). Blots are representative of 4 independent experiments with 4 different cultures.