Fig. 4: HAX-1 contributes to a reduction in c-Abl expression levels.

A Western blot analysis with specific antibodies for determination of the levels of total Flag-c-Abl status in HEK 293 cells expressing different dosages of Myc-HAX-1 protein. All cells were transfected with 1 μg Flag-c-Abl, and lanes 2, 3 and 4 were transfected with 0.5, 1.0 and 2.0 μg Myc-HAX-1, respectively. Mock (Lane 1) served as a control in which empty vector was used to transfect the cells. Cellular tubulin was used as an internal control for comparison of protein load in each lane. B Western blot analysis of HEK 293 cells transfected with the indicated plasmids. The expression of β-actin served as a loading control. C Western blot using anti-HAX-1 antibody identified the MCF-7 clones that were transfected with the HAX-1-specific siRNA or scrambled sequence. The expression level of HAX-1 and c-Abl was normalized to the tubulin loading control. D HEK 293 cells were treated with increased concentrations of H₂O₂ for 3 h and subjected to Western blot analysis. E Flag-c-Abl was co-expressed with the Myc-vector or with Myc-HAX-1 in HEK 293 cells. Cells were pulsed with [³⁵S] methionine for 45 min, washed and then incubated in [³⁵S] methionine-free DMEM for the indicated time. Lysates were immunoprecipitated with anti-Flag antibody and analyzed by SDS-PAGE and autoradiography. The half-life of Flag-c-Abl was calculated according to the intensity of the signals of three independent experiments. F Flag-c-Abl was transfected into HEK 293 cells with or without Myc-HAX-1 co-expression. Cell lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with anti-c-Cbl or anti-Flag antibody. G MCF-7 scramble cells were transfected with Myc-HAX-1, and HAX-1 knockdown cells were transfected with a Myc-HAX-1 rescue construct (Muta). For the ubiquitination assay, cells were treated with MG132 (10 μM) 12 h before harvesting. Cell lysates were immunoprecipitated with anti-c-Abl antibody or IgG as a control. The precipitates were analyzed by Western blot using indicated antibodies. H MCF-7 cells transfected with or without HAX-1 were IR treated (10 Gy) as indicated. Cell lysates were immunoprecipitated with anti-c-Abl antibody and immunoblotted using indicated antibodies. I MCF-7 scramble and two HAX-1 knockdown clones overexpressing Myc-Ub were IR (10 Gy) treated. After 2 h, cells were harvested, and cell lysates were immunoprecipitated with anti-Myc antibody. The ubiquitination levels of the precipitates were assessed by Western blot.