Fig. 7.

mDGKε-Myc is localized to the endoplasmic reticulum and to the Golgi apparatus. HEK293 cells were transfected with mDGKε-Myc, and after 48 h, cells were fixed with 4% paraformaldehyde and permeabilized with 0.05% Triton X-100 (for endoplasmic reticulum staining) or with 0.005% digitonin (for Golgi staining). A1–A3: Localization of mDGKε-Myc, (B1) STIM1, (B2) GM130, (B3) golgin-97. C1–C3: Merged images of mDGKε-Myc and the respective marker protein. Colocalized mDGKε-Myc and marker protein appear white. z-Stack images of 10 optical sections taken in the middle of a cell are shown. Insets in (C1–C3) show enlarged images of marked fragments. (A2′–A2””) Four consecutive optical sections through the Golgi showing mDGKε-Myc, (B2′–B2””) GM-130, and (C2′–C2””) merged image. (A3′–A3””) Four consecutive optical sections through the Golgi showing mDGKε-Myc, (B3′–B3””) golgin-97, and (C3′–C3””) merged images. Arrows point to Golgi fragments with intensive staining of both mDGKε-Myc and the respective marker protein. D1–D3: Reconstructed 3D images of two colocalized ROI positive for mDGKε-Myc and STIM1 (D1) or GM130 (D2) or golgin-97 (D3). Contours of the nucleus detected by Hoechst 33342 staining are shown in blue. ROI as these were used for quantitative analysis of mDGKε-Myc distribution presented in Table 1. mDGKε-Myc (magenta) was visualized with mouse anti-Myc IgG followed by donkey anti-mouse IgG-Alexa647. STIM1, GM130, and golgin-97 (green) were visualized with rabbit anti-STIM1, anti-GM130, and anti-golgin-97 IgG followed by donkey anti-rabbit IgG-FITC. Additional cells stained according to this protocol are shown in supplemental Fig. S6.