Loss of VPS45 causes accumulation of EGFR in early endosomes and inefficient degradation. (A) Serum-starved control and VPS45 KO HeLa clones were stimulated with 100 ng/mL EGF for indicated time periods, and EGFR levels were analyzed by immunoblotting. One representative experiment of 3 independent experiments is shown. (B) Cells were treated as in panel A, fixed, and costained with anti-EGFR and anti-EEA1 antibodies. Nuclei were visualized using DAPI. Enlarged images represent magnified views of boxed areas (10 × 10 µm), and profile blots along the dashed arrow (7 µm) are shown. One representative experiment of 3 independent experiments is shown. Scale bars, 10 µm. (C) Quantification of EGFR-EEA1 colocalization of data shown in panel B. Pearson correlation coefficient is plotted on the y-axis. For each genotype, ≥40 cells were analyzed. Horizontal lines indicate the means, and each symbol represents an individual cell. Statistical analysis was performed using 2-way ANOVA with Tukey multiple comparisons test. ****P < .0001.