Fig. 3.

Retrograde transport of furin does not require Vps26. HeLa cells were transfected with either control siRNA or siRNA to knockdown Vps26 for 48 hours and transfected again with FLAG–furin for a further 24 hours. (A) For immunoblotting, cells were lysed in SDS-PAGE reducing buffer and cell extracts were subjected to SDS-PAGE on a 4–12% gradient polyacrylamide gel. Proteins were transferred to a PVDF membrane and probed with rabbit anti-Vps26 antibodies using a chemiluminescence detection system. The membrane was then stripped and reprobed with mouse anti-α-tubulin antibodies. (B,C) For internalisation assays, transfected cells were incubated with mouse anti-FLAG antibodies for 45 minutes on ice. Monolayers were washed in PBS and incubated in serum-free medium for (B) 45 minutes or (C) 90 minutes at 37°C and then fixed and permeabilised. These were stained for the internalised antibody–FLAG–furin complexes with Alexa-Fluor-568-conjugated anti-mouse IgG (red) and for p230 using rabbit anti-p230 antibodies, followed by Alexa-Fluor-488-conjugated anti-rabbit IgG (green). Scale bars: 10 μm. (D) Quantification of FLAG–furin levels within the Golgi of siRNA-treated cells. The percentage (±s.e.m.) of the total FLAG–furin pixels that overlapped with p230 in each cell was determined using the plug-in OBCOL on ImageJ (n=20 for each time-point). The data from Vps26-knockdown cells is expressed as a percentage of the control siRNA data set. ***P<0.001.