VPS45 is dispensable for internalization but is required for efficient transferrin recycling. (A) Flow cytometric analysis of cargo uptake. Control and VPS45 KO HeLa clones were incubated with 32.5 µg/mL fluorescent OVA at 37°C for indicated periods, and the rate of cargo uptake was analyzed as a change in MFI from baseline (fluorescence at 4°C). Data are pooled from 2 independent experiments. Error bars indicate mean ± standard deviation (SD). Statistical analysis of significance using 2-way analysis of variance (ANOVA) followed by Tukey multiple comparisons test revealed no significant difference in OVA uptake between control and VPS45 KO HeLa clones. (B) Flow cytometric analysis of transferrin recycling in control and VPS45 KO HeLa clones. Cells were incubated with 25 µg/mL of fluorescent transferrin for 30 minutes at 37°C, followed by a chase for the indicated periods and removal of surface-bound transferrin. Representative histograms of 1 control clone and 1 VPS45 KO clone are shown. The chart shows cell-associated transferrin as the percentage of initial transferrin present at time t = 0, and pooled data from 4 independent experiments are shown. Error bars indicate mean ± SD. Statistical analysis was performed using 2-way ANOVA followed by Tukey multiple comparisons test. ns, not significant. ****P < .0001.