Fig. 3

Effect of Src homology phosphotyrosyl phosphatase 2 (SHP2) silencing on epidermal growth factor receptor (EGFR) expression. Cells were treated with chloroquine (Chl) for variable times, washed, and then immediately stimulated with epidermal growth factor (EGF) for variable times. Immunoblot analysis of EGFR in the control and SHP2-silenced (sh-2) MDA-MB-231 (a) and MDA-MB-468 (c) cells treated with 100 μg/ml chloroquine for variable times. Bar graphs show EGFR band density measurements from three independent experiments in the MDA-MB-231 (b) and MDA-MB-468 (d) cells. e Dynamics of ligand-induced EGFR degradation in the presence and absence of SHP2. After chloroquine treatment, cells were stimulated with EGF for variable times and lysates from them were analyzed for EGFR. f Bar graph shows EGFR band density measurements from three independent experiments conducted as shown in (e). g Fluorescence images of control and short hairpin RNA (shRNA)-2 cells derived from the MDA-MB-468 cells. Cells were treated with tetramethylrhodamine-labeled EGF and processed as described in Materials and methods. h Immunoblot analysis for state of EGFR ubiquitination (Ub) in control and shRNA-2 cells after stimulation with EGF for the indicated times and immunoprecipitation with anti-EGFR antibody (top panel). Bottom panel shows comparable EGFR protein levels in all lanes. i Quantitative reverse transcriptase–polymerase chain reaction on EGFR mRNA levels in the control (Con) and SHP2-silenced sh-2 cells derived from the MDA-MB-231 and MDA-MB-468 cell lines. The EGFR messenger RNA (mRNA) expression level was corrected against glyceraldehyde-3-phosphate dehydrogenase mRNA in both the control and SHP2-silenced cells. The EGFR band densities in b, d, and e were adjusted using corresponding β-actin band densities