Hook1 depletion inhibits recycling of CD98 and cell spreading in HeLa cells. (A) The amount of protein recycled back to the PM for CD98, MHCI, and the TfR in control and Hook1 siRNA-depleted cells was determined using the protocol described in Materials and methods. Cells were incubated with antibodies against CD98 or MCHI, or loaded with Alexa Fluor 594-Tf for 30 min at 37°C. After internalization, remaining surface-bound antibody was removed by a brief acid wash. Then the cells were incubated for 30 min at 37°C to allow recycling. The percentage of recycling for each cargo proteins was calculated, and shown here is the mean recycling from three independent experiments ± SD (error bars). To confirm the statistical significance of the results, the two-tailed Student’s t test was used. For CD98 recycling, the p-value for control versus Hook1-depleted cells was P < 0.05. For MHCI and TfR recycling, the values were not significantly different. (B) Control and Hook1 siRNA-treated cells were detached from the culture dish and replated onto coverslips for 6 h. The cells were fixed and stained with rhodamine phalloidin to facilitate the visualization and scoring of the phenotype. Bar, 10 µm. (C) The percentage of attached cells on the coverslips that were spread was determined by scoring >100 cells. The data shown are the means of “% cells spread” from three independent experiments ± SD (error bars).