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. 1999 Dec 7;96(25):14300–14305. doi: 10.1073/pnas.96.25.14300

Figure 3.

Figure 3

Modification of EWS/WT1 by c-Abl. (A) Immunoprecipitation of c-Abl from Jurkat cell lysates. Human c-Abl was precipitated with anti-c-Abl antibody, c-Abl(24–11) (Santa Cruz Biotechnology) and separated on an 8% SDS/PAGE. After electrophoresis, proteins were transferred onto Immobilon PVDF membrane and a Western blot was performed with the same antibody and an horseradish peroxidase-conjugated goat anti-mouse antibody (Santa Cruz Biotechnology). Molecular mass markers are provided to the left and are derived from prestained NEB protein standards (broad range). (B) In vitro phosphorylation of EWS/WT1 by c-Abl. c-Abl immunoprecipitates were resuspended with myelin basic protein (MBP) and either recombinant GST or GST-EWS/WT1 in kinase buffer [10 mM Tris⋅HCl, pH 7.5/150 mM NaCl/10 mM MgCl2/0.5 mM DTT/50 μM ATP/10 μCi γ-32P-ATP (3,000 Ci/mmol)/240 μM pervanadate] and incubated at 37°C for 1 hr with moderate agitation. Reaction mixtures were fractionated on a 10% polyacrylamide gel, dried, and exposed to X-Omat x-ray film at −70°C for 1 hr. (C) Modification of EWS/WT1 by c-Abl abolishes DNA recognition. c-Abl immunoprecipitates were resuspended with recombinant (His)6-EWS/WT1 protein in kinase buffer (10 mM Tris⋅HCl, pH 7.5/150 mM NaCl/10 mM MgCl2/0.5 mM DTT/5 mM ATP/240 μM pervanadate) and incubated at 37°C for 1 hr with moderate agitation. Reaction mixtures were analyzed by Southwestern blotting as described in Materials and Methods. The presence of c-Abl immunoprecipitates or (His)6-EWS/WT1 in the reaction mixtures is indicated (Upper). (Lower) A Western blot analysis of the recombinant EWS/WT1 after incubation with c-Abl and probed with α-C-19 antibody. (D) Phosphoamino acid analysis of in vitro-phosphorylated (His)6-EWS/WT1 by c-Abl. Phosphorylated (His)6-EWS/WT1 was separated by 10% SDS/PAGE, transferred onto Immobilon PVDF membrane, and subjected to phosphoamino acid analysis as described in Fig. 1. The positions of the unlabeled standards, detected with ninhydrine staining are shown as pS (phosphoserine), pT (phosphothreonine), and pY (phosphotyrosine).