Volume 8 issue 7 July 2007
CVAK104 Is a Novel Regulator of Clathrin-mediated SNARE Sorting
GHH Borner, AA Rana, R Forster, M Harbour, JC Smith and MS Robinson

Supplemental Figure 1. Localisation of CVAK104-GFP and CVAK104-kinase-domain-YFP in fixed HeLa cells. a, CVAK104-GFP was transiently expressed in HeLa cells. Cells were fixed, and AP-1 g and CVAK104-GFP were detected by immunofluorescence microscopy. CVAK104-GFP shows a distribution that is highly similar to endogenous CVAK104, as well as substantial colocalisation with AP-1 g (white arrowheads). b, CVAK104-kinase-domain-YFP was transiently expressed in HeLa cells. Cells were fixed, and endogenous CVAK104 and CVAK104-kinase-domain-YFP were detected by immunofluorescence microscopy. The two proteins show substantial colocalisation in vesicular structures (white arrowheads), but not in the kinase-domain-positive tubular structures.
Note that the CVAK104 antibody was raised against the C-terminal part of the protein, and therefore does not cross-react with the kinase-domain-YFP fusion protein. Scale bars = 5 μm.

Figure 1 (.jpg)

Supplemental Video 1: CVAK104-GFP expressed in HeLa cells (12x).

Video1 (mov)

Supplemental Video 2: CVAK104-GFP expressed in HeLa cells depleted of clathrin heavy chain (12x).

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Supplemental Video 3: CVAK104 kinase-domain-YFP expressed in HeLa cells (12x).

Video 3 (mov)

Supplemental Video 4 : CVAK104 kinase-domain-YFP expressed in HeLa cells depleted of clathrin heavy chain (12x).

Video 4 (mov)

Supplemental Video 5: CVAK104-GFP and Alexa Fluor 594 labeled transferrin in HeLa cells starting approximately 1 minute after beginning of transferrin uptake (250x).

Video 5 (mov)

Supplemental Video 6: CVAK104-GFP and Alexa Fluor 594 labeled transferrin in HeLa cells starting approximately 60 minutes after beginning of transferrin uptake (250x).

Video 6 (mov)

Scale bars in all movies: 10 µm.