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. Author manuscript; available in PMC: 2015 May 1.
Published in final edited form as: Mol Cancer Ther. 2014 Mar 14;13(5):1356–1368. doi: 10.1158/1535-7163.MCT-13-1021

Figure 3. Src Family Kinases mediate nEGFR translocation in TNBC.

Figure 3

(A) Constitutively active Src (caSrc) enhances nEGFR translocation in TNBC cell lines. Cells were transfected with caSrc or an empty vector control for 48 hr prior to stimulation with EGF (5 nM, 45 min) to induce nEGFR translocation. Non-nuclear and nuclear proteins were harvested. nEGFR expression was quantitated using ImageJ software (B) A negative regulator of Src, src-like adaptor protein (SLAP), blocks nEGFR translocation in TNBC cell lines. Cells were transfected with SLAP-FLAG or an empty vector control for 48 hr prior to harvesting non-nuclear and nuclear proteins. nEGFR expression was analyzed. (C) SLAP can interact with EGFR and decrease EGFR activation at Tyrosine 1101. Cells were transfected with SLAP-FLAG or an empty vector control for 48 hr prior to harvesting whole cell lysate. 250 ug of cell lysate was immunoprecipitated with an anti-SLAP antibody. The same lysate was subjected to immunoblot analysis for activation of EGFR at Tyrosine 1101. pEGFR-Y1101 activity was quantitated using ImageJ software. Inset 1: EGFR mutated at Tyrosine 1101 is deficient in nuclear localization. Vector, EGFR-WT, and EGFR-Y1101F were transfected into CHOK1 cells for 48 hr prior to stimulation with EGF (5 nM, 45 min). Non-nuclear and nuclear proteins were harvested, and nEGFR expression was analyzed.