Fig. 2: Preserved protein stability with compromised tyrosine kinase activity in EPHB4 kinase domain variants.
From: Mutation of key signaling regulators of cerebrovascular development in vein of Galen malformations
a Steady-state abundance of EPHB4 D-mis mutants. Cos-7 cells were transiently transfected with c-myc-tagged WT or EPHB4 D-mis mutants together with an eGFP-encoding vector. Numbers at top indicate percentage of GFP+ cells determined by flow cytometry (see Supplementary Fig. 3). Cells were lysed and EPHB4 abundance determined by Western blot for c-myc (left) or EPHB4 (right). Blots were probed for tubulin or beta actin (ActB) respectively to demonstrate equivalent protein loading. Numbers below indicate normalized abundance of EPHB4 D-mis mutants relative to WT EPHB4. Each experiment was repeated two times with similar results. b Stability of EPHB4 D-mis mutants. Cos-7 cells transiently transfected with c-myc-tagged WT or EPHB4 D-mis mutants were treated with cycloheximide (CHX) for the indicated times before lysis and determination of EPHB4 and ActB abundance by Western blot. Numbers indicate normalized EPHB4 abundance relative to CHX-untreated cells for each time point. Shown are the results of a single experiment. c Kinase activity of EPHB4 D-mis mutants. EPHB4 was immunoprecipitated from Cos-7 cells transiently transfected with c-myc-tagged WT or EPHB4 D-mis mutants. EPHB4 kinase activity was determined by Western blot of immunoprecipitates with an anti-phosphotyrosine antibody (pTyr). Numbers indicate normalized pTyr content of EPHB4 D-mis mutants relative to WT EPHB4. Shown are the results of one experiment of two repeats.
