Depletion of Rab22a affects CIE cargo tubular endosome biogenesis and sorting into EEA1-associated endosomes. (A) Rab22a protein levels in control or siRNA-treated cells were determined using Western blotting of cell lysates with an antibody against Rab22a and actin (loading control). (B and C) Control- or siRNA-treated cells were allowed to internalize antibody to CD147 and CD98 for 1 h at 37°C and processed for immunofluorescence and confocal microscopy. (D) Control- or siRNA-treated cells were scored for CIE cargo-containing tubules as described in Materials and methods. Results presented in the graph are the means of two independent experiments. (E) Quantitative analysis of colocalization between EEA1 and CIE cargo in control- and siRNA-treated cells. The graph shows the mean and standard deviation for the population of cells (20 cells per condition) from one representative experiment, repeated twice. Bars: (B and C) 10 µm; (B and C, insets) 5 µm.