Subcellular localization of CRAC channel variants. Stable STIM1-expressing HEK293 cells were transfected with pCAGGSM2 vectors of various constructs. Cells were stained with an anti-HA antibody and a detecting antibody conjugated with Alexa 658 and visualized via confocal microscopy (LSM510; Carl Zeiss, Inc.). Bars, 10 µm. Images show representative examples of at least 10 individual cells for each construct. (A) CRACM1-wt construct (left) and chimera of CRACM1 with the CRACM3 N-terminal region (right). Both proteins, including the chimera, are expressed in the plasma membrane. (B) CRACM3-wt construct (left) and chimera of CRACM3 with the CRACM1 N-terminal region (right). Both proteins are expressed in the plasma membrane, although the expression of the latter appears to be less. (C) CRACM1-ΔN87 (left) and CRACM1-K85E (right). Both constructs are inserted into the plasma membrane. (D) CRACM3-ΔN62 (left) has reduced plasma membrane expression, and the CRACM3-K60E construct (right) shows expression in the plasma membrane. (E) Fluorescence densities obtained from confocal images, as shown in A–C. Values represent the average fluorescent intensities normalized to the unit area of the cortical and intracellular regions of 8–15 cells each.