Figure 5. Intra-Golgi localization analysis for the role of the lumenal domain of TGN46 in CARTS-mediated export from the trans-Golgi network (TGN).

(A) Schematic representation of construct domain topology. TMD: transmembrane domain. (B) HeLa cells co-expressing the different indicated proteins (green and magenta channels) were fixed, and the localization of those proteins was monitored by fluorescence confocal microscopy. Insets correspond to zoom-in areas of the dashed, white boxed areas. (C) Pearson’s correlation coefficient between the perinuclear fluorescence signal of the x-axis indicated proteins with respect to TGN46-mRFP (empty circles) or ST-mCherry (gray circles), measured from confocal micrographs in (B). Results are from at least 10 cells from each of n = 3 independent experiments (individual values shown, with mean ± stdev; ns, p > 0.05; *p ≤ 0.05; ***p ≤ 0.001; ****p ≤ 0.0001). (D) HeLa cells co-expressing the different indicated proteins (green and magenta channels) were fixed, processed for immunostaining when required, and the localization of those proteins was monitored by fluorescence confocal microscopy. Insets correspond to zoom-in areas of the dashed, white boxed areas. (E) Pearson’s correlation coefficient between the perinuclear fluorescence signal of the x-axis-indicated proteins with respect to TGN46-mRFP (empty circles) or VSVG-HA (gray circles; detected by immunofluorescence using an Alexa Fluor 647-conjugated secondary antibody), measured from confocal micrographs in (D). Results are from at least 10 cells from each of n = 3 independent experiments (individual values shown, with mean ± stdev; ns, p > 0.05; ****p ≤ 0.0001). Scale bars in (B, D) are 10 µm.