Fig. 6. Induced phosphorylation of Grg4 upon interaction with Pax5. (A) Pax5-dependent phosphorylation of Grg4. Pax5 (where indicated) and Myc-tagged Grg4 were transiently expressed in COP-8 cells for 48 h. Grg4 was then precipitated from whole-cell lysates with a monoclonal anti-Myc antibody, incubated with λ protein phosphatase (λ-Pase) and then analyzed by Western blotting with a polyclonalanti-Myc antibody. (B) Specificity of the Grg4 phosphorylation. Myc-tagged Grg4 and Flag-tagged Pax5 or En2 proteins were co-expressed in COP-8 cells followed by Western blot analysis with anti-Myc and anti-Flag antibodies, respectively. The asterisk denotes a cross-reacting protein. (C) The phosphorylation of Grg4 depends on direct interaction with Pax2/5/8 proteins. The Pax proteins indicated were co-expressed with Myc-tagged Grg4 in COP-8 cells followed by Western blotting with polyclonal anti-Myc and anti-paired domain antibodies. (D) Phosphorylation of Myc-tagged Grg4 in NIH 3T3 and COS-7 cells co-expressing Pax5. (E) Endogenous (endo) Grg proteins are phosphorylated in Pax5-expressing COP-8 cells, as shown by Western blot analysis with a pan-TLE (Grg) antibody (Stifani et al., 1992).