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. 2015 Dec 31;113(3):644–649. doi: 10.1073/pnas.1517112113

Fig. 2.

Fig. 2.

EGFR phosphorylates FAM129B at Y593 and promotes the interaction between FAM129B and H-Ras. Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. (A) U251 cells were treated with EGF (100 ng/mL) for 15 min. (B) Purified bacterially expressed S-tagged FAM129B protein was incubated with purified active GST-EGFR (intracellular domain). A pulldown assay was performed. (C) In vitro kinase assays were performed with purified bacterially expressed WT FAM129B or FAM129B Y593F with or without GST-EGFR. (D) U251 cells expressing the indicated V5-tagged FAM129B were treated with or without EGF (100 ng/mL) for 15 min. (E) U251 cells were treated or not treated with AG1478 (1 μM) for 30 min before stimulation with or without EGF (100 ng/mL) for 15 min. (F and H) In vitro kinase assays were performed by mixing purified bacterially expressed S-tagged WT FAM129B (F and H) or FAM129B Y593F (H) with or without active GST-EGFR, which was followed by incubation with purified GST–H-Ras for a S-tag FAM129B pulldown assay. (G) U251 cells were treated with EGFR inhibitor AG1478 (1 μM) or MEK/ERK inhibitor U0126 (20 μM) for 30 min before stimulation with EGF (100 ng/mL) for 15 min. (I) U251 cells overexpressing the indicated V5-tagged FAM129B were treated with or without EGF (100 ng/mL) for 15 min.