Fig. 5.

Furin requires the TGN golgin GCC185 for retrograde transport. HeLa cells were transfected with either control siRNA or siRNA against mRNA encoding GCC185 for 48 hours and transfected again with FLAG–furin for a further 24 hours. (A) Endogenous GCC185 was stained with affinity-purified rabbit anti-GCC185 antibodies, followed by Alexa-Fluor-568-conjugated anti-rabbit IgG. 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 affinity-purified rabbit anti-GCC185 antibodies using a chemiluminescence detection system. The membrane was then stripped and reprobed with mouse anti-α-tubulin antibodies. For internalisation assays, transfected cells were incubated with mouse monoclonal anti-FLAG antibodies for 45 minutes on ice. Cells 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. Monolayers were stained for the internalised antibody-bound FLAG–furin 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 GCC185-knockdown cells. The percentage 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 GCC185-knockdown cells are expressed as a percentage (±s.e.m.) of the control siRNA data set. **P<0.01; ***P<0.001.