Depletion of Hook1 affects tubular endosome formation and endosomal sorting of CIE cargo proteins. HeLa cells were treated with On-TARGET plus SMARTpool siRNA directed against Hook1 as described in Materials and methods. (A) Western blot showing the reduction of Hook1 protein levels in siRNA-treated versus control cells. Hook1 was detected using rabbit anti-Hook1 antibody and Alexa Fluor 680–conjugated anti–rabbit antibody. Actin was used as a loading control. (B and C) Control and Hook1 siRNA-depleted cells were incubated with CD147, CD98, and MHCI antibodies for 1 h at 37°C. After internalization, the cells were fixed and processed for immunofluorescence. Insets show enlarged views of the boxed regions. (D) Quantitative analysis of the percentage of cells exhibiting tubular endosomes loaded with CD147, CD98, and MHCI in control versus Hook1 siRNA-depleted cells. Data presented is the mean of three independent experiments ± SD (error bars). (E) The percentage of colocalization between CIE cargo and EEA1 after Hook1 depletion was determined using MetaMorph software (see Materials and methods). The graphs shows the mean and standard error for the population of cells (error bars; 20 cells per condition) from one experiment, repeated three times. Bars: (B and C) 10 µm; (B and C, insets) 5 µm.