Figure 5.

Pull-down of membrane proteins using anti-Rab11 antibodies. Rab11-containing microsomes from rat kidneys were partially purified using differential and density-gradient centrifugation (see Materials and methods). These fractions were incubated with anti-Rab11 antibody, and the antibody–membrane complexes were captured with protein G–coated beads. (A) Significant amounts of Rab11 were pulled down from the starting material (“input”) when labeled with anti-Rab11 but not with a control antibody. The bands at 55 kD represent the IgG species used for labeling. (B) Significant amounts of γENaC were pulled down from the starting material when labeled with anti-Rab11 but not with control antibody. Both full-length (80 kD) and cleaved (60–65 kD) species were detected. (C) Na depletion increased the amount of cleaved γENaC in Rab11-containing vesicles. The 55-kD bands represent the anti-Rab11 IgG used for the pull-down. The results are representative of four independent experiments. Quantification is shown in Fig. 7.