ARF6 activation and transferrin endocytosis in bone marrow cells.
(A) Protein lysates were prepared from Smap1+/+ and Smap1–/– bone marrow cells and incubated with GST or GST-GGA1 coupled to glutathione-Sepharose. The bound fraction was processed for immunoblot detection by anti-ARF6–specific and anti-panARF antibodies, as indicated. An asterisk represents nonspecific bands. The amounts of ARF6 in each lysate prior to incubation with GST or GST-GGA1 were also evaluated by immunoblotting (see “Total ARF6”). (B and C) Bone marrow cells were prepared from Smap1+/+ and Smap1–/– mice and labeled with fluorescein-transferrin at 4°C. Excessive transferrin in the medium was washed away (initially bound transferrin at this time is shown as “4°C” as indicated in the top left of B), and, after incubation of cells at 37°C for the indicated time, surface-remaining transferrin was stripped off. Cells were labeled with PE-anti-Ter119 and processed for flow cytometry. The Ter119+ fraction was gated, and the transferrin-derived fluorescence intensities are displayed. Relative amounts of internalized fluorescein were measured by comparing fluorescence intensities at 0 minutes and each given time. Cells were prepared from 3 independent pairs of Smap1+/+ and Smap1–/– mice and processed for assays. Averages ± SD of internalized transferrin were calculated for each incubation time at 37°C (n = 3). *P < 0.05.