Fig. 4

ER-to-GA VSVg trafficking is affected in human fibroblasts with TSC1 knockdown. a Western blot analysis of TSC1, phospho-S6 (P-S6), and phospho-AKT (P-AKT) levels in protein lysates from control pLKO and shTSC1 fibroblasts. b Quantification of Western blot analysis of hamartin, P-AKT, and P-S6, normalized to tubulin, in protein lysates that were obtained as in (a). *p < 0.05 (one-sample t-test). N = 3 independent experiments. c Representative time-lapse confocal images of living fibroblasts: control (pLKO) and with TSC1 knockdown (shTSC1). The fibroblasts were electroporated with Str-li_VSVGwt-SBP-EGFP. Trafficking of VSVg-EGFP was visualized using the RUSH assay for 90 min after the addition of biotin (time 0). Scale bar = 20 µm. d Quantitative analysis of the experiments performed as in (c). The graph presents VSVg-EGFP fluorescence intensity in the Golgi apparatus region at each time point, normalized to the maximum value. The data are expressed as the mean for all of the analyzed cells. Error bars indicate SEM. N = 3 independent experiments. Number of cells per variant (n): pLKO (25), shTSC1 (35). e Comparison of VSVg-EGFP fluorescence intensity in the Golgi apparatus region at 32 min (left panel) and 54 min (right panel). The data are expressed as the mean for all of the analyzed cells. Error bars indicate SEM. *p < 0.05 (Mann–Whitney test). The number of independent experiments and analyzed cells per variant are the same as in (d). f Comparison of VSVg-EGFP maximum accumulation times in the Golgi apparatus region. The data are expressed as the mean for all of the analyzed cells. Error bars indicate SEM. *p < 0.05 (Mann–Whitney test). The number of independent experiments and analyzed cells per variant are the same as in (d)