Figure 2. Changes in retrograde transport kinetics of CDMPR to the TGN upon silencing Vps26, Rab9a, or TIP47.

(A, B, C, D) Cells stably expressing EGFP-CDMPR were transfected with non-targeting siRNA (control-kd) or with siRNA silencing expression of Vps26 (A) or Rab9a (C) as described in Fig 1. The cells were labeled with [³⁵S]sulfate for up to 75 min in the presence of 2 μg/ml VHH-2xTS. The nanobodies were isolated by Ni/NTA beads and subjected to SDS-gel electrophoresis followed by Western-blot (WB) analysis (anti-His6) and autoradiography ([³⁵S]). In parallel, aliquots of the cell lysates were immunoblotted for actin as a control for the amount of cells used. Experiments as shown in panels (A) and (C) were quantified in panels (B) and (D), respectively, and presented as the percentage of the value of control-kd cells after 75 min (mean and SD of three independent experiments; two-sided t test: *P < 0.05; **P < 0.01). Control-kd is shown as black squares and target-kd as gray circles; uptake as open symbols, sulfation as filled symbols. (E, F) HeLa cells stably expressing His6/myc-tagged cathepsin D or PAUF were transfected with non-targeting control siRNA or RNAs silencing expression of Vps26 or Rab9a. Cells were incubated in serum-free medium supplemented with 5 mM mannose-6-phosphate for 2 h. Secreted procathepsin D (pCatD) or PAUF were collected by Ni/NTA beads and analyzed by immunoblotting with anti-myc antibodies. (G, H) Missorted procathepsin D or PAUF was quantified from immunoblots as shown in panels C and E, respectively, and normalized to the values of control knockdown cells (mean and SD of four [pCatD] and three [PAUF] independent experiments). (I, J) Cells stably expressing EGFP-CDMPR were transfected with non-targeting siRNA (control-kd) or with siRNA silencing expression of TIP47 and assayed and quantified as described above in panels (A, B, C, D) (mean and SD of three independent experiments).