Cell Culture and Expression Vectors:
293 cells and mouse embryonic fibroblasts (MEF) were maintained in Dulbecco's modified Eagle’s medium supplemented with fetal calf serum (10%) at 37°C and 5% CO2. A mammalian expression vector encoding TAB2 (pCMV-HA-TAB2) was described previously (Takaesu et al., 2000
). The TAB2ΔCUE mutant was generated by utilizing EcoRV site to remove sequences encoding amino acids 1–53 of TAB2 cDNA. TAB2(F20A) and TAB2(E20D) mutants were generated by PCR and verified by DNA sequencing. The expression vector for ubiquitin (pCDNA3.1-Myc-Ub) was a kind gift from Dr. Keiji Tanaka (The Tokyo Metropolitan Institute of Medical Science).
Antibodies and Immunoprecipitation:
Polyclonal rabbit antibodies to TRAF6, TAK1, TAB1, and TAB2 have been described previously (Ninomiya-Tsuji et al., 1999
; Takaesu et al., 2000
). Polyclonal rabbit antibodies to JNK, p38, IκBα, IKKα and monoclonal antibodies to Myc and ubiquitin were purchased from Santa Cruz. Monoclonal antibodies to phospho-JNK and phospho-p38 were purchased from Cell Signaling. Monoclonal antibody to HA (HA. 11) was purchased from Covance. For immunoprecipitation, MEFs were plated (1 x 106
) on 10 cm dishes 24 hrs prior to stimulation. Cells were starved in serum-free medium at 37°C for 3 hrs and stimulated with IL-1β (Roche). After stimulation, cells were washed once with ice-cold phosphate-buffered saline (PBS) and lysed in 0.5% Triton X-100 lysis buffer containing 20 mM HEPES (pH 7.4), 150 mM NaCl, 12.5 mM β-glycerophosphate, 1.5 mM MgCl2, 2 mM EGTA, 10 mM NaF, 2 mM DTT, 1 mM sodium orthovanadate, 1 mM PMSF and 20 mM aprotinin. Cellular debris was removed by centrifugation at 10,000 x g for 5 min. Proteins from cell lysates were immunoprecipitated with 1 μg of various antibodies and 15 μl of protein G-Sepharose (Pharmacia). For the transfection studies, 293 cells (1 x 106
) were plated in 10 cm dishes, and transfected by the calcium phosphate precipitate method with a total of 15 μg DNA of various expression vectors . After incubation for 48 hrs, cells were lysed with 0.5% Triton X-100 lysis buffer.
Reporter Gene Assay: For the reporter gene assays, 293 cells (1.6 x 105 cells/well) were plated into 6-well (35 mm) plates. At 24 hrs after plating, cells were transfected with an Ig-κ-luciferase reporter plasmid and the indicated expression plasmids. A plasmid containing the β-galactosidase gene under the control of the β-actin promoter (pAct-β-Gal) was used for normalizing transfection efficiency.
Yeast two-hybrid analysis: The yeast strain PJ69-4A was transformed with yeast expression vectors encoding proteins fused in-flame to the Gal4 DNA-binding domain (GBD) or the Gal4 activation domain (GAD). The plasmid pACT2-wtUbi for expressing wild-type ubiquitin fused with Gal4-activation domain was a kind gift from Dr. Linda Hicke (Northwestern University). The interactions between the bait and prey proteins were evaluated by growth on medium lacking histidine and containing 3 mM 3-AT (histidine synthesis inhibitor).
Subcellular localization: HeLa cells, plated on chamber slides, were transfected by Transfast transfection reagent (Promega). After transfection at 36 hrs, cells were washed with PBS, fixed in 4% paraformaldehyde, and washed 2 times with PBS. The fixed cells were subsequently permeabilized with acetone for 2 min at room temperature and blocked with 10% serum for 30 min. The cells were then incubated with various combinations of anti-HA, anti-Flag, anti-IKKα, anti-TAB2 and anti-TAK1, followed by Cy2-conjugated anti-mouse and/or Cy3-conjugated anti-rabbit secondary antibody (Amersham), and examined by fluorescent microscopy (Olympus).