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. Author manuscript; available in PMC: 2022 Sep 17.
Published in final edited form as: Matrix Biol. 2021 Sep 17;103-104:1–21. doi: 10.1016/j.matbio.2021.09.001

Figure 2: Serum treatment induces ligand-independent EphA2 phosphorylation.

Figure 2:

Human VSMCs were transfected with either mock or siRNA targeted against EphA2 for 24 hours. Cells were plated onto Matrigel coated plates overnight in either serum-free or 1% serum. Protein was isolated and analyzed via immunoblotting (A) Representative immunoblots and (B) densitometry analysis of phospho-Y772, phospho-S897, and total EphA2. Total EphA2 was normalized to GAPDH, pEphA2 Y772 and pEphA2 S897 were normalized to total EphA2. Values expressed as fold change from baseline (mock 0’). C) Human VSMCs were serum-starved for four hours, treated with 1μg/mL Fc-EphrinA1 for 5 or 30 minutes, followed by immunoblotting for phosphorylated (Y588, Y772, and S897) and total EphA2. D-E) Human VSMCs were seeded onto Matrigel-coated glass coverslips overnight in 1% serum-containing media and stained for (D) S897-phosphorylated EphA2 (teal), (E) Y772-phosphorylated EphA2 (teal), (D,E) Y397-phosphorylated FAK (red), and DAPI (nuclei). F) Cells were plated onto Matrigel-coated glass slides overnight in 1% serum, followed by integrin adhesion isolation and protein expression was measured via immunoblot. G) EphA2 KO mouse aortic VSMCs cells were transfected with EphA2-WT, K646M, or R103E, plated overnight in 1% serum, and stained for phospho-EphA2 Y772 (teal), Y397-pFAK (pink), EphA2 (white) and DAPI (nuclei). n=4. Data are expressed as mean ±SEM. Statistical comparisons were made using 2-way ANOVA with Bonferroni post-test. A p-value less than 0.05 is considered significant.