Figure 3

Digestion of ABCA1 on the cell surface by trypsin and release of ECD fragments into the medium. (A) BHK/ABCA1-207HA cells after induction with mifepristone were treated with 50 μg/mL of trypsin for 0, 10, 30, or 60 min at 37 °C. Triplicate experiments are shown. The cells without mifepristone induction were also treated with trypsin for 60 min. After trypsin was inactivated, the medium was collected and slot blot analyses of HA-positive peptides in the medium were performed. Similar experiments were performed three times and the representative result was shown. (B) Relative intensities of HA-positive signals were calculated. The average values (arbitrary units) are shown with S.D. (C) BHK/ABCA1 cells were treated with 50 μg/mL of trypsin for 0, 10, 30, or 60 min at 37 °C. After trypsin was inactivated, surface proteins were biotinylated at 4 °C. Biotinylated proteins (8 μg) were precipitated with monomeric avidin agarose resin and analyzed by western blotting with NB400-105. Full-length ABCA1 and produced fragments are indicated by white and black arrowheads. The average values (relative to the value at 0 min) are shown at the bottom. (D) BHK/ABCA1-MM cells were treated with 50 μg/mL of trypsin for 0, 10, 30, or 60 min at 37 °C. After trypsin was inactivated, surface proteins were biotinylated at 4 °C. Biotinylated proteins (8 μg) were precipitated with monomeric avidin agarose resin and analyzed by western blotting with NB400-105. Full-length ABCA1 is indicated by a white arrowhead. The average values (relative to the value at 0 min) are shown at the bottom. (E) Schematic diagram of the full-length ABCA1. Recognition sites of two antibodies, NB400-105 and KM3110, are shown (not to scale). The original images of Fig. 3C,D are in Supplemental Fig. 4.