Fig. 5. Retrograde transport of TGN38 in cells expressing ARFRP1 mutants. (A) HeLa cells co-expressing FLAG-TGN38 and either HA-ARFRP1(WT), HA-ARFRP1(Q79L) or HA-ARFRP1(T31N) were incubated with mouse monoclonal anti-FLAG M2 antibody on ice for 50 minutes, washed, and further incubated at 37°C for 60 minutes. The cells were then fixed and stained with rat anti-HA and rabbit anti-ß-COP antibodies followed by Alexa Fluor 488-conjugated anti-rat, Cy3-conjugated anti-mouse and Cy5-conjugated anti-rabbit secondary antibodies. Bar, 20 µm. (B) The efficiency of the retrograde transport of TGN38 was estimated by calculating the ratio of the perinuclear overlapped area of the TGN38- and ß-COP-positive structures versus the total area of the perinuclear ß-COP-positive structures. In each case, the areas were estimated for over 10 cells.