Sigma-Aldrich (Saint Louis, MO). FuGENE 6 was purchased from Roche Molecular Biochemicals (Indianapolis, IN). Lipofectamine 2000 was purchased from Invitrogen. Anti-HA Y-11 and Anti- MEKK1 were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). Anti-phosphorylated c-Jun, anti-c-Jun, anti-phosphor- ylated ERK1/2 and anti-ERK1/2 were purchased from Cell Signaling Technologies (Beverly, MA). Anti-HA was purchased from Covance (Cumberland, VA). Anti-b tubulin, aflag (M2), aflag-conjugated agarose beads and EZ view HA-conjugated agarose beads were obtained from Sigma-Aldrich (Saint Louis, MO). Protein G sepharose fast flow 4 was purchased from GE Healthcare Biosciences (Piscataway, NJ). The phospho-MEKK1 antibody was generously donated by Dr. Ewen Gallagher. This antibody has been shown to recognize active MEKK1 [15,23]. The CaspACE 3 assay was obtained from Promega (Madison, WI). FITC secondary antibodies were purchased from Caltag (Burlingame, CA) and Texas red-phalloidin from Molecular Probes. The goat anti-rabbit IgG horseradish peroxidase was obtained from (Santa Cruz Biotechnology, Inc). Cell culture and transfections HEK 293T cells were maintained with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, and 100 mg/ml streptomycin. Transient transfections were performed using FuGENE 6 according to the manufacturer instructions. DT40 and murine B cell lines were maintained in DT40 medium (RPMI 1640 medium containing 10% FBS, 1% chicken serum, 50 mM 2-mercaptoeth- anol, 50 U of penicillin per ml, and 50 mg of streptomycin per ml) at a density of 0.56106 to 1.56106 cells/ml. HeLa cells were maintained in DMEM with 5% FBS and 100 IU/ml penicillin and 100 mg/ml streptomycin. Creation of murine B cell line A murine stem cell virus vector containing the BCR-Abelson fusion gene and GFP was transfected with helper virus plasmid into HEK 293T cells, and supernatant was harvested after 36 hours. The filtered supernatant was used to infect bone marrow cells from either wild type or MEKK1-deficient mice from the lab of Bing Su. After approximately three weeks B cell populations arose and clones were selected, as previously described [38]. MEKK12/2 reconstitution Viral transduction was used to reconstitute DT40 MEKK1- deficient cells; briefly, HEK 293T cells were transfected with a viral helper construct plus pBABEpuro containing the indicated MEKK1 construct, using standard calcium phosphate methods. MEKK1- deficient cells were then infected with the filtered 293T cell supernatants followed by selection with 0.4 mg/ml of puromycin. Western blotting and immunoprecipitation MEKK1 and MAPK western blotting was previously described [2]. Briefly, 56106 cells were lysed in modified RIPA or lysed directly in 16 SDS loading buffer. Samples were sonicated, and 25 mg or 75 mg of protein were boiled in SDS loading dye and run on a 10% or 7% polyacrylamide electrophoresis separating gel respectively for MAPK or MEKK1. The protein was then transferred onto nitrocellulose membrane and then probed with the indicated antibody. For immunoprecipitation of DT40 cell lines, 506106 cells were resuspended in lysis buffer (20 mM Tris- Hcl pH 7.5, 150 mM NaCl, 1% Triton-X 100 and 2 mM EDTA supplemented with protease inhibitors, 5 mM NaVO4 and 1 mM NaF). Cells were sonicated and lysates were cleared by MEKK1-Dependent Apoptosis Requires the PHD centrifugation. 20 ml of HA-agarose beads were added to the lysates and incubated overnight at 4uC. Samples were then washed four times with lysis buffer, boiled in SDS loading buffer, run on SDS-PAGE gel and protein was detected by anti-phosphorylated or total MEKK1 antibody. For immunoprecipitation from transiently transfected 293T cells, 20 ml of flag-conjugated beads were added to cell lysates instead of HA-agarose beads. Analysis of Apoptosis DNA laddering assays were performed as previously described [2]. Briefly, 3.756106 wild type DT40 or MEKK1-deficient cells were treated with 1 mM vinblastine sulfate, 2 mg/ml cytochalasin D or 90 nM okadaic acid and then processed as previously described. Propidium iodide staining protocol was followed as previously described [39]. In brief, cells were stimulated with 1 mM vinblastine sulfate, 2 mg/ml cytochalasin D or 90 nM okadaic acid for the indicated time points, washed once with PBS and resuspended in Nicoletti buffer (0.1% sodium citrate and 0.1% Triton-X 100) and fluorescence intensity was analyzed via flow cytometry. In the caspase 3 activity assay, 26107 cells treated for the indicated times with 1 mM vinblastine sulfate, 2 mg/ml cytochalasin D or 90 nM okadaic acid, were lysed in 50 ml of ice-cold cell lysis buffer containing 50 mM HEPES (pH 7.4), 100 mM NaCl, 0.1% 3-[(3-cholamidopropyl)-dimethylammonio]- 1-propanesulfonate (CHAPS), 1 mM dithiothreitol, and 0.1 mM EDTA, flash frozen in an ethanol-dry ice bath, and stored at 280uC before use. Caspase 3 activity assay experiments were performed as described in the Calbiochem protocol for colori- metric caspase 3 substrate I (Ac-DEVD-pNA). A405 readings were taken on a plate reader at four hour intervals. Immunofluorescence To assess MEKK1 localization, HeLa cells were used due to superior surface adhesion and high transfection efficiency. HeLa cells were grown on coverslips and transfected using FuGENE6 according to the standard protocol. Twenty four hours post transfection cells were fixed with cold methanol for three minutes or 4% paraformaldehyde for 12 minutes, washed 3 times with PBS and then permeabilized with 0.1% Triton-X 100 in PBS for 3 minutes. Primary antibodies were used at a 1:300 dilution in PBS with 3% BSA overnight at 4u and washed 3 times with PBS for 5 minutes. Secondary antibodies were conjugated to either FITC or TRITC and used at a dilution of 1:1000 in PBS with 3% BSA. CIP treatment DT40 cells were treated with 1 mM vinblastine for six hours and cells were lysed in modified RIPA. MEKK1 was immunoprecip- itated with 4 mg aMEKK1C-22 and 40 ml protein G sepharose beads. Beads were resuspended in NEB buffer 3 with or without calf alkaline phosphatase and incubated for one hour at 37uC. SDS loading buffer was added and reactions run on SDS-PAGE. Membranes were probed with aMEKK1. Acknowledgments We thank Bing Su for providing the Mekk12/2 and wild type bone marrow used to create murine B cells. We thank O. Witte for the p210 BCR-ABL vector and helper plasmid used to transform bone marrow cells into murine B cells. Author Contributions Conceived and designed the experiments: ET AA GC. Performed the experiments: ET. Analyzed the data: ET AA GC. Contributed reagents/ materials/analysis tools: GYC EG RK. Wrote the paper: ET AA GC. PLoS ONE | www.plosone.org 11 February 2011 | Volume 6 | Issue 2 | e17310