Fig. 3.

Interactions between the nucleotide sugar transporters SLC35A2, SLC35A3 and SLC35A4 (abbreviated A2, A3 and A4, respectively). a FRET analyses of A4-mVenus interactions with mCherry-A2, mCherry-A3 and mCherry-A4. The experiments were done mainly as described for Fig. 1, except that we used MGAT2-mVenus and mCherry-AE2 pair as a negative control. A statistical significance was assigned using one-way ANOVA with Tukey’s Post Hoc test (± SD, n = 3, ***p < 0.001). b BiFC analyses of the same interactions. After cell transfections with the depicted BiFC enzyme constructs, BiFC signals were quantified for both single (negative control) and double transfectants using Operetta High-Content Imaging System. Mean mVenus fluorescence signal was quantified in the Golgi region by averaging the signal from at least 10,000 VN and VC-positive cells. The bars denote data values after subtracting the mean mVenus fluorescence intensity in negative control areas (single transfectants) from those obtained with double transfectants. Statistical significance was calculated using one-way ANOVA with Tukey’s Post Hoc test, (***p < 0.001). c Immunofluorescence detection of the BiFC signal with a confocal microscopy. After the transfections, VC-positive cells were visualized using rabbit anti-FLAG primary antibody and anti-rabbit Alexa Fluor 594 secondary antibody (red) with an appropriate filter set. VN-positive cells were counterstained with mouse anti-HA primary antibody and anti-mouse Alexa Fluor 405 secondary antibody and specific filter set for Alexa 405 (cyan). BiFC signal was detected using the YFP filter set (green). Bars = 10 µm