Fig. 4

ITM2A interacts and is phosphorylated by HUNK. (a) Coomassie brilliant blue staining showed that the peptide “VAIKVIDKKRAKKDTYVTKNLRREGQIQQMI” existed in the 70 kDa–100 kDa band. (b) Co-immunoprecipitation showed the interaction between ITM2A and HUNK in HEK293T cells. (c) Flag-HUNK were obtained from the immunoprecipitate in HEK293T cell lysates using Flag antibody. The Flag-HUNK was incubated with the in vitro purified GST-ITM2A WT or T35A protein at 30 °C for 30 min with or without ATP addition, and then the reaction products were subjected to western blotting. (d) Flag-ITM2A was obtained from the immunoprecipitate in SKBR-3 cell lysates with or without HUNK knockdown using Flag antibody. Total phosphorylated threonine was detected in the western blotting assay. (e) SKBR-3 cells transfected with HA-ITM2A or Flag-HUNK were starved for the indicated times, and HA-ITM2A was immunoprecipitated using HA antibody and used for western blotting analysis. Purified Flag-HUNK was used in the in vitro kinase assay using purified MBP protein as a substrate. (f) SKBR-3 cells were transfected with wild-type ITM2A and its T35A mutant simultaneously with or without HUNK siRNA co-transfection for 48 h. Cell lysates were obtained and used for detection by western blotting with the indicated antibodies. (g) HEK293T cells were transfected with empty vector and Flag tagged HUNK simultaneously with or without ITM2A siRNA co-transfection for 48 h. Cell lysates were obtained and used for detection by western blotting assay with the indicated antibodies