Figure 2

Enrichment of autophosphorylated Mirk/Dyrk1B consistent with the protein expression may be mediated by activated ERK1/2. (A), the averaged spectral counts corresponding to the phosphotyrosine (pY) autophosphorylation site of Mirk/Dyrk1B in NSCLC cells were analyzed by LC-MS/MS proteomics. (B), the pY bands of Mirk/Dyrk1B were detected by Western blot analysis in four lines H292, H358, A549 and H1299 after the cell protein extracts were immunoprecipitated with Mirk antibody and then immunobloted by pY monoclonal antibody. (C), H292 cells treated with/without U0126 (10 μM) for 48 h or 10% FBS for 24 h were collected and the protein expressions of Mirk (69 and 71 Kda), ERK1/2 and P-ERK1/2 (42/44 kDa), as well as alterations of cyclin D1 (36 Kda) and p27kip1 (27 kDa) were analyzed by western blot. Equal loading and transfer were shown by repeat probing with β-actin (42 kDa). NSCLC, non-small cell lung cancer; LC-MS/MS, liquid chromatography coupled to tandem mass/mass spectrometry; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; IP, immunoprecipitate; WB, western blot.