FIGURE 8:

Knockdown of VPS33A and UVRAG, but not STX17, impairs the endocytic pathway. (A) HeLa cells treated with siRNA against luciferase, VPS33A, STX17, or UVRAG were serum starved overnight and stimulated with 100 ng/ml EGF. The expression level of EGFR was determined at indicated time intervals. Arrows indicate a degradation product of EGFR. Short exp., short exposure; Long exp., long exposure. Protein levels of EGFR after 0, 0.5, 1, 2, and 4 h of EGF stimulation were quantified by densitometry. Quantification of three independent experiments is shown in the graph. *p < 0.05 compared with the luciferase siRNA–treated group, one-way analysis of variance followed by least significant difference post hoc tests. (B) siRNA-transfected HeLa cells were serum starved overnight and stimulated with 100 ng/ml Alexa 647–conjugated EGF for 1 h. Then cells were rinsed, fixed, and stained with antibodies against EEA1 and LAMP1. Scale bars, 10 μm. Quantification of the number of double EEA1- and EGF-positive or double LAMP1- and EGF-positive structures as a percentage of total EGF-positive puncta is shown in the graph. Data are presented as mean ± SEM values of 10 different cells.