Figure S2.

In resting Jurkat cells, STIM1 and Orai1 are located in the ER and PM, respectively. (A and C) Confocal images of cellular distribution of endogenous STIM1 and Orai1 in Jurkat cells (A) and PBMCs (C). PM and ER of cells (first row) were stained with CellMaskOrange and ER-tracker red, respectively. Images shown as false color in blue. The second row shows immunostaining of STIM1 (green) and Orai1 (magenta) and secondary antibody tagged with Alx488 and Alx647, respectively. An overlay of both channels is shown in right column. (B and D) Line plots for each marker were taken in positions report in merged images. Fluorescence intensity of either Orai1/PM (B) or STIM1/ER (D) were normalized to the highest value of each signal; the colors of line plots correspond to those in images. All scale bars, 10 μm. The antibodies for detecting STIM1 and Orai1 are specific. (E) Jurkat cells in which Orai1 was knocked out (Fig. S2) generate no more signal in immunostaining with Orai1 antibody, while still producing signal with STIM1 antibody. (F) Staining of WT Jurkat cells in which either the primary STIM1 or Orai1 antibodies (top row) or the respective secondary antibodies (bottom row) were left out generated no appreciable fluorescent signals.