Fig. 3: The HAX-1 and c-Abl interaction leads to increased c-Abl tyrosine kinase activity.

A HEK 293 cells were co-transfected with Flag-c-Abl and Myc-HAX-1 or Myc-vector. Cell lysates were immunoprecipitated with anti-Flag antibody, and the immunoprecipitates were normalized by c-Abl level and immunoblotted with indicated antibodies. B In vitro immune complex kinase assay. Flag-c-Abl and Flag vectors were transfected with or without Myc-HAX-1 into HEK 293 cells as indicated. Proteins were purified using anti-Flag antibody-coupled Sepharose beads and then eluted with Flag peptide. In the kinase assay reaction system, the purified recombinant fusion proteins were incubated with GST-Crk fusion protein or GST protein at 30 °C for 30 min. ATP was added to the reaction buffer as indicated. The products were analyzed by SDS-PAGE and Western blot with indicated antibodies. C MCF-7 scramble and HAX-1 knockdown cell lines (MCF-7/siHAX-1) were transfected with Flag-Abl vector. Cell lysates were immunoprecipitated with anti-Flag antibody and analyzed by Western blot with indicated antibodies. The immunoprecipitates were normalized by c-Abl level. D The MCF-7 scramble or HAX-1 knockdown cell line (MCF-7/siHAX-1) was transfected with Myc-HAX-1 or vector control. Before being harvested, cells were treated with the indicated dosage of H₂O₂ for 3 h. The cell lysates were analyzed by immunoprecipitation and immunoblotted. The immunoprecipitates were normalized by c-Abl level. E The MCF-7 scramble or HAX-1 knockdown cell line (MCF-7/siHAX-1) were subjected to 10 Gy irradiation. Cell lysates were immunoprecipitated with anti-c-Abl antibody, and immunoblots were probed with indicated antibodies. The immunoprecipitates were normalized by c-Abl level. F Flag-c-Abl was co-transfected with Myc-HAX-1 or Myc-HAX-1(Q190X) in 293 cells. Cell lysates were immunoprecipitated with anti-Flag antibody. The immunoprecipitates were normalized by c-Abl level and immunoblotted with indicated antibodies. G HEK 293 cells were transfected with Myc-HAX-1, Myc-HAX-1(Q190X) or Myc-vector. Cell lysates were immunoprecipitated with anti-c-Abl antibody. The immunoprecipitates were normalized by c-Abl level and immunoblotted with indicated antibodies. H MCF-7 cells transfected with Myc-HAX-1 or Myc-vector plasmids were incubated with target primary antibodies from two different species or with anti-c-Abl antibody as a control. The red spots reveal c-Abl/HAX-1 interaction. Nuclei are stained with Hoechst 33342 (left). Quantification of HAX-1-c-Abl interaction complexes. The number of complexes per cell was counted in at least three fields. Quantifications were given as the mean±S.D. Representative results are shown from experiments repeated three times (right). ***p < 0.001, Student’s t test.