Fig. 5

The critical role of DAB2IP in modulating IR-induced ASK1-JNK pathway activation. A EC109-shluc and EC109-shDAB2IP cells were initially treated with 3 Gy dose of IR, and 36 h later, the indicated protein levels were evaluated by Western blotting. Silencing DAB2IP by lentivirus obviously increased the protein levels of p-JNK. No changes were observed in p-ERK and p-AKT levels. B DAB2IP enhanced IR-induced dephosphorylation of ASK1-pSer966 and JNK activation. Kyse150 cells were initially transfected with pcDNA3.1-DAB2IP or vehicle pcDNA3.1 plasmid, followed by treatment with or without IR, and 36 h later, ASK1-pSer966, and JNK activation were measured by Western blotting. C EC109 cells were firstly infected with DAB2IP-shRNA or control luc-shRNA lentivirus, and subsequently received with or without IR. After 36 h, Western blotting was performed to detect levels of ASK1-pSer966 and p-JNK. Results are representative data of three independent experiments. D DAB2IP enhanced IR-induced disruption of ASK1-14-3-3 complex, but not ASK1-Trx complex. Kyse150 cells were initially transfected with pcDNA3.1-DAB2IP or vehicle pcDNA3.1plasmid, followed by treatment with or without IR, and 36 h later, cell extracts were firstly immunoprecipitated by anti-ASK1 antibody, and then the precipitate was immunoblotted (IB) with 14-3-3 and Trx antibody. Input was equivalent to 30% of the cell lysate used in the immunoprecipitation. E EC109 cells were initially infected with lentivirus carried with DAB2IP-shRNA or luc-shRNA, subsequently received with or without IR. After 36 h, interaction of ASK1 with endogenous 14-3-3 or Trx was determined by coimmunoprecipitation with primary anti-ASK1 antibody followed by Western blot with 14-3-3 or Trx antibody. The input represents 30% of the cell lysates. Each immunoprecipitation experiment was repeated at least three times and a representative experiment is shown. F The enhanced activity of DAB2IP on IR-activated JNK signal was dependent on the presence of ASK1. The Kyse150 cells were firstly infected with ASK1-shRNA or control luc-shRNA lentivirus, and then each group cells were transfected with pcDNA3.1-DAB2IP or vehicle pcDNA3.1 plasmid. All group cells were followed by 3 Gy IR treatment, and 36 h later, JNK activation were evaluated by Western blotting with p-JNK antibody. Arrows point to the bands representing indicated proteins. The representative blots of three independent experiments were shown