The rabbit polyclonal Mirk/Dyrk1B antibody (C-term, AP7538b) was purchased from Abgent (San Diego, CA, USA). Anti-p27kip1, anti-cyclin D1, anti-ERK1/2, and goat anti-mouse IgG horseradish peroxidase (HRP)-conjugated secondary antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-poly (ADP-ribose) polymerase (PARP) and anti-phosphotyrosine (pY) purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-C-Raf and -phosphorylated C-Raf (P-C-Raf), anti-phosphorylated ERK1/2 Threonine 202/Tyrosine 204 (P-ERK1/2) were purchased from BD Biosciences PharMingen (San Diego, CA, USA). Anti-β-actin and donkey anti-rabbit IgG HRP-conjugated secondary antibody were purchased from Sigma (St. Louis, MO, USA) and Amersham Biosciences (Piscataway, NJ, USA), respectively.
Cell lines and cell culture
Human ovarian cancer cell lines used were OV2008, OVCAR3, OVCAR5, SKOV3, MDAH2774, OVCAR10, OV1063, and OVCAR8. Of eight cell lines, SKOV3 and OVCAR3 were purchased from American Type Culture Collection (Manassas, VA, USA); others and all NSCLC cell lines used in this study, such as HCC827, PC-9, H1975, H292, H358, H441, A549, and H1299 were gifts from H. Lee Moffitt Cancer Center and Research Institute, USA. All lines were maintained in DMEM supplemented with 10% heat-inactivated (56°C, 30 minutes) fetal bovine serum (FBS; Invitrogen, Grand Island, NY, USA). Monolayer cultures were incubated at 37°C in a 95% humidified atmosphere air containing 5% CO2.
Small interfering RNA treatment
Cells were reverse transfected with small interfering RNAs (siRNAs) using lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer’s instructions. The Mirk/Dyrk1B siRNA duplexes as well as the corresponding nonspecific control siRNA duplexes as described  were supplied by Dharmacon (Pittsburgh, PA, USA). For combined treatment, cells were pretreated with U0126, an inhibitor of MEK purchased from CalBiochem-NovaBiochem Corporation (La Jolla, CA, USA) at dose escalation for 1 h followed by combination with a constant 20nM dose of siRNAs. Through indicated duration of each treatment, cells treated were harvested and saved for the following experiments.
Cell proliferation assay
Cells were plated in 96-well plates, and siRNA transfection was performed for 72 hours as described above. Cellular proliferation was measured by [3-(4,5)-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) analysis . Briefly, after cells were washed with PBS, they were incubated in MTT solution for 4 hours and then supplemented with 100 μl of dissolving solution (10% SDS in 0.01 M HCl). The absorbance (optical density units) was measured with a microplate spectrophotometer (Bio-Rad Laboratories, Hercules, CA, USA) with Microplate Manager 5.1 software at wavelengths of 590 nm and 660 nm. Each assay was performed in quadruplicate.
Flow cytometry analysis
After 72-hour treatment with siRNAs, cells were subjected to flow cytometry analyses of apoptosis. Apoptosis was assayed using Pharmingen PE-conjugated monoclonal active caspase-3 antibody apoptosis kit without modification as described previously . We determined the percentage of cells in G1, S, and G2/M by propidium iodide staining as described previously . A total of 10,000 cells per experimental condition were used for processing and analysis of fluorescence on Becton-Dickinson FACScan (BD, Franklin Lakes, NJ, USA) using CellQuest software.
Western blot analysis
Cells were washed twice with cold PBS and lysed with buffer A [10 mM Tris–HCl (pH 7.4), 1% Triton X-100, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 5 μg/ml aprotinin]. After incubation for 30 minutes on ice, the suspensions were centrifuged (15,000 g for 30 minutes). The supernatants were removed and stored at −80°C until analysis using gel electrophoresis. The protein concentration was determined by Bio-Rad protein estimation assay according to the manufacturer’s instructions. For Western blot analysis, ~60-100 μg of whole cell proteins were separated using 10% or 12% SDS-PAGE and transferred to nitrocellulose membranes. After blocking of the membranes with 10 mM Tris–HCl (pH 7.4), 150 mM NaCl, and 0.1% Tween 20 containing 5% nonfat dry milk at room temperature for 60 minutes, the membranes were incubated with indicated antibodies at 4°C overnight and then with the HRP-conjugated secondary anti-rabbit or anti-mouse antibodies at room temperature for 60 minutes. Each protein was detected using the enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ, USA) system. β-actin was used as an internal control.
Immunoprecipitations were performed with 500 μg of whole cell protein lysates, using Protein A-agarose (Roche, Indianapolis, IN, USA). Briefly, equal amount of protein lysates were incubated with Mirk/Dyrk1B antibody and normal rabbit IgG used as negative control. After incubation for overnight at 4°C, the immune complexes were precipitated with Protein A-agarose. The immunoprecipitates were washed with lysis buffer according to the manufacturer’s instructions, then separated by SDS-PAGE, and transferred to nitrocellulose membranes followed by incubation of pY or Mirk antibodies for western blot analysis as described above.
Phosphopeptide immunoprecipation and analysis by liquid chromatography coupled to tandem mass/mass spectrometry (LC-MS/MS)
Phosphopeptide immunoprecipitation for eight NSCLC cell lines: HCC827, PC9, H1975, H292, H358, H441, A549, and H1299 was performed using phosphoscan kit (P-Tyr-100, Cell Signaling) according to the manufacturer’s instructions. Using an immunoaffinity peptide profiling technique, Mirk/Dyrk1B unique phosphopeptides with sites associated with Mirk/Dyrk1B protein were isolated and quantitated by LC-MS/MS proteomics analysis as described previously . Results were subjected to sequest-IPI database searching according to criteria specified by molecular and cellular proteomics/cell signaling technology (MCP/CST). Duplicate samples for each cell line each with two technical runs were then filtered according to a 80% peptide identification probability and a 50% protein identification probability.
Each experiment was repeated three times. Data are presented as mean ± SD. Statview 5.0 software was used for statistical analyses. Statistical comparison among the groups was performed using one-way analysis of variance (ANOVA), followed by the Fisher least significant difference test. The correlations between Mirk/Dyrk1B expression and active ERK1/2 were analyzed by simple regression. Differences were considered to be statistically significant when P was less than 0.05.