Figure 4
DGKδ triggers ER release of IFT88-containing COPII vesicles. (A) Representative immunostaining images of endogenous IFT88 and SEC13 in DGKδ−/− and their matching control (DGKδ+/+) MEFs. (B) Calculation of colocalization co-efficient in (A). (C) Proximity ligation assay for and (D) quantification of the interaction between IFT88 and SEC13 in DGKδ −/− MEFs and the control (DGKδ+/+) MEFs. Data are means plus standard deviation and the statistical significance was calculated with the Student T test, n > 40. (E) Representative images and (F) quantification of ER areas in DGKδ+/+ and DGKδ−/− MEFs. The cells were marked by exogenously expressed DsRed-ER, and the white dotted lines mark the contour of the cell. Data are means plus standard deviation and the statistical significance was calculated with the Student T test, n > 20. (G) Real-time qPCR analysis of Hspa5 mRNA in DGKδ+/+ and DGKδ−/− MEFs. (H) Westernblot analyses Endo H sensitive of VSVgts045 in DGKδ+/+ and DGKδ−/− MEFs. The cells were routinely maintained at the permissive 37 °C, shifted to the nonpermissive 40 °C for 24 hours to let GFP-VSVgts045 accumulate in the ER after transfection with a plasmid vector expressing GFP-VSVgts045, then added cycloheximide to inhibit protein synthesis and shifted back to permissive 32 °C for various time periods to monitor VSVgts045 trafficking after adding cycloheximide to inhibit protein synthesis. At the end of each time point, the cells were harvested for Endo H digestion and analyzed by westernblot. (I) Western blot results showing the distribution of ERGIC53, IFT88 and Sec13 in COP II-coated vesicles which budding in vitro. And the distribution of the three proteins and β-actin were taken as the control. (J) Bar graph depicts the ratio of relative intensity of ERGIC53, Sec13 and IFT88 in budding and total portions in DGKδ+/+ and DGKδ−/− MEFs in (I). (K) Iodixanol density gradient sedimentation of calnexin, IFT88, SEC13, and exogenously expressed HA-DGKδ in DGKδ+/+ and DGKδ−/− MEFs. **P < 0.01, ***P < 0.001.
