Volume 8 issue 6 June 2007
The TGN golgin, GCC185, is required for endosome to Golgi transport and maintenance of Golgi structure
Merran C. Derby¹, Zi Zhao Lieu¹, Darren Brown², Jennifer L. Stow², Bruno Goud3 and Paul A. Gleeson^1#

Figure S1: Distribution of EEA1 and LAMP1 is unaffected in GCC185-depleted cells. HeLa cells transfected with miRNA-1 for 96 h were fixed with 4% paraformaldehyde and stained with (A) monoclonal anti-EEA1 antibodies, followed by Alexa-conjugated anti-mouse IgG and (B) monoclonal anti-LAMP1 antibodies, followed by Alexa-conjugated anti-mouse IgG. Endogenous GCC185 was detected with rabbit anti-GCC185 antibodies followed by Alexa-conjugated anti-rabbit IgG. Bar = 10 µm

Figure 1 (jpg)

Figure S2: Detection of surface E-cadherin in GCC185-depleted cells. HeLa cell monolayers were transfected with control miRNA (lacking GFP) for 96 h or miRNA-1 (lacking GFP) for 72 or 96 h and transfected a second time with E-cad-YFP 24 h prior to staining.    Cell monolayers were fixed with 4% paraformaldehyde and stained with monoclonal anti-E-cad antibodies followed by Alexa Fluor 568-conjugated goat ant-mouse IgG.  Fixed monolayers were then permeabilised and stained with human anti-p230 antibodies followed by Alexa Fluor 647-conjugated goat anti-human IgG. .  Bar = 10 µm

Figure 2 (jpg)

Figure S3: Anterograde trafficking of VSVG in GCC185-depleted cells. HeLa cell monolayers were transfected with miRNA-1 (lacking GFP) for 96 h and transfected a second time with VSV-G-GFP 24 h prior to fixation.   Cell monolayers were stained with monoclonal anti-GM130 antibodies followed by Alexa-conjugated goat anti-mouse IgG.   Shown are represented examples of transfected cells. Bar = 10 µm

Figure 3 (jpg)

Figure S4: GPP130 is localised to Golgi fragments in GCC185-depleted cells. HeLa cells transfected with miRNA-1 for 96 h were fixed with 4% paraformaldehyde and permeabilised and stained with rabbit anti-GPP130, followed by Alexa-conjugated anti-rabbit IgG and monoclonal anti-GM130 antibodies, followed by Alexa-conjugated anti-mouse IgG.  Bar = 10 µm or 5 µm (magnified images).

Figure 4 (jpg)

Figure S5: STxB does not accumulate in early endosomes in GCC185-depleted cells. HeLa cells transfected with miRNA-1 for 96 hours were incubated with Cy3-conjugated STxB for 45 minutes on ice and incubated at 37 ºC for five hours, followed by fixation with 4% paraformaldehyde.  Cells were stained with monoclonal anti-EEA1 antibodies followed by Alexa -conjugated anti-mouse IgG. Boxed images in a GFP+ transfected cell is shown magnified in lower panel.  Bar = 10 µm or 5 µm (magnified images).

Figure 5 (jpg)