Inhibition of ERC-to-MVBs/SGs transport in RBL-Syt IX⁻ cells by a GTPase-deficient mutant of ARF1. (A) RBL-Syt IX⁻ cells were cotransfected with HA-TGN38 and either ARF1-GFP (i-iii) or ARF1(Q71L)-CFP (iv-vi) cDNA and grown on glass coverslips for 24 hours. Cells were labeled with monoclonal anti-HA antibodies followed by Cy3-conjugated donkey anti–mouse IgG. Bars equal 5 μm. The arrow points to a cell double-transfected with HA-TGN38 and ARF1(Q71L)-CFP. (B) RBL-Syt IX⁻ cells were double-transfected with HA-TGN38 and ARF1(Q71L)-CFP cDNA and grown on glass coverslips for 48 hours. Cells were then incubated with monoclonal anti-HA antibodies (2 μg/mL) for 30 minutes at 4°C, washed, and subsequently incubated at 37°C for 30 minutes. Cells were labeled with Cy3-conjugated donkey anti–mouse IgG. Bars represent 8 μm. The phase-contrast image is shown on the left.