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Fig. 7. VSVG-GFP transport in cells expressing ARFRP1 mutants. (A) HeLa cells coexpressing GFP-tagged VSVG tsO45 and either HA-tagged ARFRP1(WT), ARFRP1(Q79L) or ARFRP1(T31N) were incubated at 40°C overnight and then shifted to 32°C for 30 minutes or 1 hour, and stained with rat anti-HA antibody followed by Cy3-conjugated anti-rat IgG. Asterisks indicate cells expressing ARFRP1-HA. (B) HeLa cells were transfected as described in A and incubated at 40°C overnight and then shifted to 32°C for 1 hour. The cell surface proteins were then biotinylated as described in Materials and Methods. Biotinylated surface proteins recovered with streptavidin-agarose beads (top panel) and total cell lysates (one twentieth amount of input; middle and bottom panels) were subjected to immunoblotting with anti-GFP (top and middle panels) or anti-HA antibody (bottom panel). (C) The band intensity in the top panel in B was estimated by Image Gauge software. This is representative of two independent experiments.





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