Figure 3. STIM2 translocates to ER-PM junctions following ER Ca2+ depletion and regulates Orai1.
(A–C) YFP-STIM2 was expressed (~24 hour) in HeLa (A), HUVEC, (B), and HEK293T (C) cells and confocal images were taken before and 2 minutes after 1 μM thapsigargin addition.
(D) Comparison of the distribution of CFP-STIM1 and YFP-STIM2 constructs 2 min after addition of thapsigargin.
(E) Ca2+-binding deficient YFP-STIM2 (point mutation in EF-hand) is prelocalized to ER-PM junction sites and does not alter its localization after Ca2+ store depletion.
(F) Knockdown of the PM Ca2+ channel Orai1 significantly reduces the increase in basal Ca2+ resulting from STIM2 expression. HeLa cells were transfected for two days with GL3, Orai1, Orai1, or Orai3 siRNA and then transfected for 24 hours with YFP-STIM2 or YFP as control. Cells used in the analysis expressed YFP at 7.5 to 15 fold above background. N=10 sites.