FIGURE 3.

Positive feedback loop between EGFR and USP13. A, USP13 and EGFR levels were determined in PC9 cells transfected with either empty vector or pEF‐FLAG‐USP13, and treated with afatinib or DMSO (−) 24 hours posttransfection. Cells were lysed 48 hours posttransfection. β‐Actin was used as a loading control. B, USP13 and EGFR mRNA levels in PC9 cells reverse‐transfected with NT or USP13 siRNAs 2 and 4. Cells were lysed 24 hours posttransfection. C, As in (A) using PC9 cells reverse‐transfected with non‐targeting (NT) or USP13 siRNAs 2‐4. Cells were lysed 96 hours posttransfection. β‐Actin was used as the loading control. D, EGFR degradation rates were determined in the presence of cycloheximide (CHX) (40 μg/mL, 1 hour pretreatment). PC9 cells were also treated with spautin‐1 or DMSO for 72 hours (left graph), or NT or USP13 siRNA B (right graph) for 96 hours. EGFR and β‐actin levels were quantified, and EGFR values normalized to the β‐actin (loading control) values. Graphs show mean ± SEM. E, USP13 mRNA levels were determined using qRT‐PCR in PC9 cells 24 hours posttreatment with afatinib or DMSO. F, Levels of indicated proteins were determined in reverse‐transfected PC9 cells with NT or USP13 siRNAs 2 and 4. Cells were treated with afatinib or DMSO (as indicated) for 48 hours and lysed 96 hours posttransfection. β‐Actin was used as the loading control [Color figure can be viewed at wileyonlinelibrary.com]