Fig. 4

The c.333del p.(Ser112Profs*115) variant affects protein levels of COPII components and intracellular trafficking. (A) The Golgi morphology and the localization of GOSR2 (membrin) are not altered in fibroblasts from individual V-8. Representative confocal images of fibroblasts fixed and stained with antibodies recognizing the endogenous membrin/GOSR2 (green) and the Golgi marker ManII (red). Scale bars represent 25 μm. (B) Representative immunoblot analysis of lysates prepared from control (CTRL) and V-8 fibroblasts and probed for SEC24C, SEC23B, SEC13, SEC31A, SAR1, and tubulin (as a loading control). Molecular size standards are shown to the right of the blots. N = 3. (C) The RUSH assay was performed to assess the traffic between the ER and the Golgi. Representative images from the movies used for the quantification are shown at the indicated time points. Scale bars represent 25 μm. (D) Cells were imaged every 2 minutes after the addition of biotin to release the cargo molecule ST-eGFP from the ER and quantified for control (CTRL) and V-8 fibroblasts. Graph shows normalized ST-eGFP fluorescence in Golgi region (arbitrary units). (E) Surface expression of the GPI-anchored proteins CD55, CD59, and CD73 was assessed by flow cytometry using antibodies specific to each protein and correcting for the background using isotype-specific antibodies. The absolute mean fluorescence intensity (MFI) of samples is shown ± SD, in triplicates. Student’s t test was performed to show the data significance level.