Fig. 5: Rafts depletion induces endogenous EGFR-PTPH1 interaction, EGFR dephopshorylation, and its intracellular arrest in MDA-MB-468 TNBC cells. | Oncogenesis

Fig. 5: Rafts depletion induces endogenous EGFR-PTPH1 interaction, EGFR dephopshorylation, and its intracellular arrest in MDA-MB-468 TNBC cells.

From: NOTCH3 inactivation increases triple negative breast cancer sensitivity to gefitinib by promoting EGFR tyrosine dephosphorylation and its intracellular arrest

Fig. 5

a Immunofluorescence assay (IF) was performed by using anti-EGFR (green) and anti-GM1 (red) antibodies to reveal the endogenous EGFR-rafts colocalization, shown in yellow (merge). Nuclei were DAPI labeled (blue). b Raft (R) and non-raft (NR) fractions derived from Methyl-β-cyclodextrin (MβCD)-treated and untreated cells were used for immunoblot assay with anti-pEGFR(Y1173) (indicated as pEGFR) and anti-EGFR antibodies, to test activated and total EGFR expression in rafts compartment, respectively. Anti-transferrin and anti-GM1 antibodies were used as a fraction markers. c Cells have been activated with EGF ligand for the times indicated, in the presence or absence of MβCD: the expression of phospho-EGFR at tyrosine 1173 and 1068 residues and total EGFR was determined in whole cell extracts by immunoblot analysis using the specific indicated antibodies. d–f MDA-MB-468 cells were treated with MβCD and stimulated with EGF for 60 min: control or anti-PTPH1 antibody immunoprecipitates were probed with anti-EGFR, to detect the EGFR-PTPH1 binding, and with the anti-PTPH1 antibody, to show PTPH1 immunoprecipitated protein levels. The inputs indicated in the panel shows 5% of each total lysate d. Relative EGFR extracellular expression (EGFREC) was evaluated by FACS e. IF assay was performed by using anti-EGFR (red) antibody to reveal the endogenous EGFR intracellular localization. Nuclei were DAPI labeled (blue). White arrows indicated peri-nuclear EGFR localization in EGF stimulated MβCD-treated cells (f). a, f Representative single plane confocal IF images captured using a × 60 oil objective. Scale bar: 10 μm. In both b and c, western blotting against the anti-β-actin was used as a loading control. All data are representative of at least three independent experiments, each in triplicate. Results shown in e are expressed as the means average deviations and P-values were calculated using Student’s T-test (i.e., ns, not significant P > 0.05, **P ≤ 0.01)

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