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. 2016 Aug 29;214(5):555–569. doi: 10.1083/jcb.201602057

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© 2016 Jungas et al.

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Figure 4. — CitK is tyrosine phosphorylated by Src in response to EphB2 signaling. (a) HeLa cells were transfected with EphB2-HA and either mock stimulated or stimulated with Efnb1-Fc. Protein lysates (top, Extract) were immunoblotted with antibodies against CitK and HA. Actin was used as loading control. Endogenous CitK was immunoprecipitated (bottom, Ip) and immunoblotted with P-Tyr, CitK, and HA antibodies. (b) HEK-293 cells were cotransfected with EphB2 and either myc-tagged CitK FL or myc-tagged Cit-N and stimulated with Efnb1-Fc for 20 min. Protein lysates (top, Extract) were immunoblotted with antibodies against Myc, EphB2, and P-EphB (EphB^Y594). Tubulin was used as a loading control. Exogenous CitK was immunoprecipitated (bottom, Ip) with an anti-Myc antibody and immunoblotted with Myc and P-Tyr antibodies. (c) Increasing amounts of purified His-Myc–tagged CitK produced in HEK-293 cells were incubated with recombinant EphB2 kinase domain in the presence of ATP. Negative controls (two lanes on the right) are samples without EphB2 recombinant kinase. Samples were analyzed by immunoblotting with antibodies against Myc and P-Tyr (top). No tyrosine phosphorylation on CitK was detected. Autophosphorylation of recombinant EphB2 kinase (bottom) indicates that the kinase was active. (d) HEK-293 cells were cotransfected with HA-tagged EphB2 and Myc-tagged CitK FL as indicated and either mock treated or pretreated with PP2 or SU6656 before stimulation with Efnb1-Fc for 20 min. Protein lysates (Extract, top) were immunoblotted with antibodies against Myc, HA, Src, and activated Src (Src^P-Y416), as indicated. Tubulin was used as loading control. Exogenous CitK was immunoprecipitated using an anti-Myc antibody and immunoblotted using P-Tyr and anti-Myc antibodies (Ip, bottom). (e) Increasing amounts of purified His-Myc–tagged CitK were incubated with recombinant Src in the presence of ATP. The negative control (right lane) is a sample without recombinant Src. Samples were analyzed by immunoblotting with Myc, Src, and P-Tyr antibodies. Purified CitK is phosphorylated on tyrosines in the presence of recombinant Src. Bottom panels show that recombinant Src kinase is autophosphorylated. (f) U251 cells were transfected with control siRNAs or siRNAs targeting Src and mock stimulated or stimulated with Efnb1-Fc. The proportion of U251 cells connected by an ICB was quantified. These experiments were performed at the same time as the experiments presented in Fig. 1 c; thus the controls (si-ctl) are the same. Error bars correspond to SEM. Statistical p-value is indicated when significant. ns, nonsignificant.