Fig. 4: The loss of Synj1 leads to intracellular accumulation of transferrin in HeLa cells.

(a-h) Internalisation assay of Alexa-488-conjugated transferrin (Tf) in Ctli and Synj1i HeLa cells (see also Materials and methods section). Briefly, Tf was added to the cells at 37 °C for 7 min (pulse; a, d). After washing out, cells were incubated in culture medium for different indicated times (chase; b, c, e, f). Representative single confocal sections a-f show that Tf is uptaken similarly in control and silenced cells, whereas it is more intracellularly stalled in Synj1-interfered cells. Bars, 10 μm. Higher magnification pictures are shown in the insets. 3D reconstructions are shown in Supplementary Figure S6a-f. g Mean fluorescence intensity (arbitrary unit, a.u.) of Tf is shown. Experiments were performed three independent times in different silenced cells (pool2 and cl1 for sh-1; pool1 for sh-2). Error bars, means ± SD; n ≥ 50 cells, **p < 0.01, Student’s t-test. (h) Curves of Tf internalisation expressed as mean values of fluorescence measured at chase times compared with the fluorescent signal after pulse (set to 1) are shown (p < 0.001, Bonferroni test after significant ANOVA). i, j Immunodetection of Tf receptor at the surface upon Tf induction by biotinylation assay (see Materials and methods section) in Ctli and Synj1i cells. Briefly, cells were incubated with Tf for 30 min at 4 °C to prevent its internalisation (time 0), washed and then warmed at 37 °C in culture medium. At the end of each chase time, cells were labelled with LC-biotin. One-tenth of lysates (total) was kept before streptavidin precipitation. Densitometric analysis is shown j, and the results were expressed as percentage of the amount of protein at the time 0. Error bars, means ± SD; **p < 0.01, Student’s t-test