Fig. 8.

Retrograde transport of FTT is Rab9 independent and Vps26 dependent. (A) HeLa cells were transfected with either control siRNA, Rab9#1 siRNA or siRNA to knockdown Vps26 for 48 hours and transfected again with FLAG–FTT for a further 24 hours. Monolayers were incubated with mouse anti-FLAG antibodies for 45 minutes on ice. Cells were washed in PBS and incubated in serum-free medium for 45 minutes at 37°C and then fixed and permeabilised. Monolayers were stained for the internalised antibody-bound FLAG–FTT with Alexa-Fluor-568-conjugated anti-mouse IgG (red) and for p230 with rabbit anti-p230 antibodies followed by Alexa-Fluor-488-conjugated anti-rabbit IgG (green). Scale bar: 10 μm. (B–D) Quantification of FLAG–FTT levels within the Golgi of siRNA-treated cells. The percentage of the total FLAG–FTT pixels that overlapped with p230 in each cell was determined using the plug-in OBCOL on ImageJ (n=20 for each sample). Data are expressed as a percentage (±s.e.m.) of the control siRNA data set. ***P<0.001.