Fig. 4

Inactive Rab4b blocks retrograde trafficking to the TGN. A Western blots of Rab4b on lysates prepared from cells transfected with (ctrl: 30 nM) or anti-Rab4b siRNA (5, 30 nM) as described in [27]. Quantification of Rab4b mRNA expression in cells treated with 20 nM ctrl or anti-Rab4b siRNA. B Western blot of sortilin as in Fig. 3A from control- or Rab4b siRNA-treated cells, and quantification of 3 experiments. *, p < 0.05 (two-way ANOVA). (C) Western blots of CI-M6PR, sortilin, and tubulin as loading control from controlor Rab4b siRNA-treated cells incubated without or with leupeptin and E64 and the quantification of 3 independent experiments. Significance compared to the ctrl/no leu/E64 condition with * p < 0.05, ** p < 0.01, *** p < 0.001; significance compared with the Ctrl within the condition no leu/E64 and leu/E64 ^# p < 0.05 (one-way ANOVA). D Western blots of sortilin, and tubulin as loading control, from control or Rab4b siRNA treated wt cells or Rab4b overexpressing cells. E Quantification of the percentage of iCD8-M6PR at the Golgi in randomly acquired whole fields in control- or Rab4b siRNA-treated cells using the protocol 1 (Additional file 1: Fig. S2A). F I colocalization index (RColoc) between iCD8-CI-M6PR and Golgi, or iCD8-CI-M6PR and co-internalized Tf, determined in the same experiments as in E, using the colocalization plugin of the open ImageJ software [51]. G Quantification the percentage of iCD8-M6PR at the Golgi in randomly acquired whole fields in control- or Rab4b siRNA-treated cells using the protocol 2 (Additional file 1: Fig. S2A) and representative images on the left panel. H Quantification of iCD8-M6PR in the Golgi of single cells overexpressing HA-Rab4b S22N (red star marks) and in the surrounding non-transfected cells (NT) and representative images according to protocol 1 (Additional file 1: Fig. S2A). E–H Significance compared to siRNA ctrl condition with *p < 0.05, **p < 0.01, ****p < 0.0001 (Student t test). Bars are for 10 µm