Files in this Data Supplement:
Table S1. Comparison of the lengths of the transmembrane domains of proteins sorted in the endocytic pathway.
Fig. S1. Endosome-to-TGN transport of furin does not involve the Rab11-positive recycling endosome. (A) HeLa and (B) CHO cells were co-transfected with GFP-Rab11 (green) and FLAG-furin for 24 hours and incubated with rabbit anti-FLAG antibodies for 45 minutes on ice. Thereafter, cells were washed in PBS and incubated in serum-free media for 15 minutes or 45 minutes at 37°C, as indicated, and then fixed and permeabilised. Cells were stained for internalised antibody-FLAG-furin complex with Alexa-Fluor-568-conjugated anti-rabbit IgG (red). (B) GM130 was stained using mouse monoclonal anti-GM130 followed by Alexa-Fluor-647-conjugated anti-mouse IgG (pseudo-coloured green). (C) The number of FLAG-furin pixels that overlapped with GFP-Rab11 in B was expressed as a percentage of the total number of FLAG-furin pixels within each cell using the plugin OBCOL on ImageJ program (n=20 for each time-point) as described in Materials and Methods. Scale bar: 10 µm.
Fig. S2. Furin does not require SNX1 for transport to the TGN. HeLa cells were transfected either with control siRNA or SNX1 siRNA for 48 hours and transfected again with FLAG-furin for a further 24 hours. (A) Cells were fixed and permeabilised and stained for SNX1 using mouse monoclonal anti-SNX1 antibodies followed by Alexa-Fluor-568-conjugated anti-mouse 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 mouse anti-SNX1 antibodies using a chemiluminescence detection system. The membrane was then stripped and reprobed with mouse anti-α-tubulin antibodies. (B-D) For internalisation assays, transfected cells were incubated with rabbit anti-FLAG antibodies for 45 minutes on ice. Cells were washed in PBS and incubated in serum-free media 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-rabbit IgG (red) and for GM130 using mouse monoclonal anti-GM130 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 SNX1 siRNA treated cells. The percentage of the total FLAG-furin pixels that overlapped with GM130 in each cell was determined using the plugin OBCOL on ImageJ (n=20 for each time-point). The data from SNX1 siRNA treated cells is expressed as a percentage of the control siRNA data set.
Fig. S3. Furin does not require SNX2 for transport to the TGN. HeLa cells were transfected with either control siRNA or SNX2 siRNA for 48 h and transfected again with FLAG-furin for a further 24 hours. (A) Cells were fixed and permeabilised and stained for SNX2 using mouse monoclonal anti-SNX2 antibodies. 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 mouse anti-SNX2 antibodies using a chemiluminescence detection system. The membrane was then stripped and reprobed with mouse anti-α-tubulin antibodies. (B) For internalisation assays, transfected cells were incubated with rabbit anti-FLAG antibodies for 45 minutes on ice, washed in PBS and incubated in serum-free media for 45 minutes at 37°C and then fixed and permeabilised. Monolayers were stained for the internalised antibody-bound FLAG-furin with Alexa-Flour-568-conjugated anti-rabbit IgG (red) and for GM130 using mouse monoclonal anti-GM130 antibodies, followed by Alexa-Fluor-488-conjugated anti-mouse IgG (green). Scale bar: 10 µm. (C) Quantitation of FLAG-furin levels within the Golgi of SNX2 siRNA treated cells. The percentage of the total FLAG-furin pixels that overlapped with GM130 in each cell was determined using the plug-in OBCOL on ImageJ (n=20 for each time-point). The data from SNX2 siRNA treated cells is expressed as a percentage of the control siRNA data set.
Fig. S4. Furin is dependent on SNAREs implicated in the late endosomal but not early endosomal-to-TGN transport. HeLa cells were transfected with control siRNA and syntaxin-6 siRNA (A), syntaxin-10 siRNA (B) or syntaxin-16 siRNA (C) for 48 hours and transfected again with FLAG-furin for a further 24 hours. Monolayers were subjected to internalisation assays as described in supplementary material Fig. S3. The percentage of the total FLAG-furin pixels that overlapped with GM130 or p230 in each cell was determined using the plugin OBCOL on ImageJ (n=20 for each sample). The data from cells treated with (A) syntaxin-6 siRNA, (B) syntaxin-10 siRNA or (C) syntaxin-16 siRNA are expressed as a percentage control siRNA data set. **P<0.01, *P<0.05.
Fig. S5. The early-endosomal-to-TGN trafficking of TGN38 requires Vps26 and SNX2. HeLa cells were transfected either with control siRNA, Vps26 siRNA, SNX1 siRNA, SNX2 siRNA, Rab9#1 siRNA, GCC185 siRNA or syntaxin-10 siRNA for 48 hours and transfected again with TGN38-CFP for a further 24 hours. Monolayers were incubated with monoclonal anti-TGN38 antibodies on ice for 30 minutes. Cells were then washed in PBS and incubated in serum-free medium at 37°C for 60 minutes or 120 minutes and then fixed and permeabilised. The internalized antibody-bound TGN38 was stained with Alexa-Fluor-568-conjugated anti-rabbit or anti-mouse IgG and anti-GM130 or anti-p230 antibodies. Levels of antibody-bound TGN38 pixels within the Golgi region of each sample were determined. The percentage of the total antibody-bound TGN38 pixels that overlapped with GM130 or p230 in each cell was determined using the plugin OBCOL on ImageJ (n=20 for each sample). The data from (A) Vps26 siRNA, (B) SNX1 siRNA, (C) SNX2 siRNA, (D) Rab9#1 siRNA, (E) GCC185 siRNA or (F) syntaxin 10 siRNA treated cells is expressed as a percentage of the control siRNA data set. *P<0.05; NS, not significant.
Fig. S6. Intracellular trafficking of FTF. HeLa cells were transfected with FLAG-tagged FTF construct for 24 hours and incubated with anti-FLAG antibodies for 45 minutes on ice followed by a wash with PBS. Monolayers were either fixed and permeabilised (0 min) or incubated in serum-free media for 45 minutes or 60 minutes at 37°C and then fixed and permeabilised. Monolayers were stained for the internalised antibody-bound FLAG-FTF with Alexa-Fluor-568-conjugated anti-rabbit IgG (red) and for (A) GM130 using mouse monoclonal anti-GM130 antibodies (green) or (B) CD63 using mouse monoclonal anti-CD63 antibodies (green). Scale bars: 10 µm.
