EphA3 kinase in LK63 cells is inhibited by strong PTP activity. (A) EphA3 tyrosine phosphorylation in α-EphA3 immunoprecipitates (IPs) of whole cell lysates from LK63 or EphA3/HEK293T cells stimulated for indicated times with preclustered ephrin-A5-Fc was examined with α-phosphotyrosine (PY) and α-EphA3 antibodies. (B) In vitro kinase activity was assessed in α-EphA3 IPs from nonstimulated (−) cells, subjected to stringent washes prior to incubation with exogenous ATP (+) and α-PY Western blot analysis. IPs from lysates of nonstimulated (−) or ephrin-A5-Fc–stimulated (+) cells are analyzed for comparison. (C) EphA3 was depleted by immunoprecipitation from lysates of ephrin-A5-Fc–stimulated cells; α-EphA3 IPs from LK63 cells were incubated (20 minutes) in EphA3-depleted cytosolic fractions of EphA3/HEK293 cells (reverse IP) and vice versa. The tyrosine phosphorylated EphA3 from these in vitro kinase assays was compared with the in vivo phosphorylation levels in the corresponding IPs from both cell types. α-EphA3 Western blots of parallel samples indicate that similar quantities of EphA3 were analyzed in all samples. (D) EphA3 levels in the cytosolic fractions used in the kinase assays were determined by Western blot analysis before and after immunodepletion.