Figure 5

Quantification of ABCA1 on plasma membrane of BHK/ABCA1 cells. (A) 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, cells were lysed, total cellular proteins (4 μg) were resolved on SDS-polyacrylamide gels, and ABCA1 was detected by western blotting with NB400-105. Full-length ABCA1 is indicated by a white arrowhead. The average values of full-length ABCA1 (relative to the value at 0 min) are shown at the bottom. Vinculin is shown as a loading control. (B) Quantitative comparison of purified ABCA1-GFP and ABCA1 in whole-cell lysates. Proteins were resolved on 5–20% gradient SDS-polyacrylamide gels, and ABCA1 was detected by western blotting with NB400-105. Experiments were performed in duplicate, and representative data are shown. (C) Standard curve obtained from Fig. 4B. Because band intensity seemed to be saturated at 2.20 × 10⁻¹⁴ mol of ABCA1, the standard curve was generated from data obtained with lower amounts of protein. Relative intensities, normalized to the intensity at 2.20 × 10⁻¹⁴ mol of ABCA1, are shown. Experiments were performed in duplicate, and representative data are shown. The original images of Fig. 5A,B are in Supplemental Fig. 4.