Chemicals and antibodies
Hydroxyurea (HU) was dissolved in ddH2O at 1 M stock concentration and filter-sterilized. MG-132 (Calbiochem) was prepared as 10 mM stock solution in DMSO and added to cells at 10 mM final concentration 30min before additional treatments. The antibodies to EXO1 (rabbit polyclonal F15) and MSH6 (mouse monoclonal 66H6) were previously described. Additional antibodies used in this study were purchased from Santa Cruz Biotechnology: goat polyclonal to OMNI (sc-499G), mouse monoclonal to MDM2 (sc-965); Sigma: mouse monoclonals to Flag (F3165) and HA (12CA5); NeoMarkers: mouse monoclonal to EXO1 (ab4, clone 266). Anti-mouse and anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies were from GE-Healthcare.
Recombinant proteins expression and purification
Recombinant EXO1 and MutSα (MSH2/MSH6) were expressed and purified as described in and respectively. Plasmids pET28a+-Flag-KAP1 and pET28a+-His-MDM2 were introduced by electroporation into the E. coli strain BL21 (DE3). Protein expression was induced by 1 mM IPTG at 37°C for 4 h. The cell pellet (20 g) was resuspended in 50 ml of ice-cold buffer A (50 mM Hepes, pH 8.0, 300 mM NaCl, 10 mM imidazole, 0.1% Triton X-100, protease complete-inhibitor cocktail (Roche), 0.1 mM phenylmethylsulfonyl fluoride, PMSF), sonicated, and cleared by ultracentrifugation at 4°C. The resulting supernatant was applied onto a 1 ml Ni-NTA column (GE Healthcare). The column was developed with 20 ml gradient of 10–500 mM imidazole in buffer A. Peak fractions were pooled, diluted 1:10 in buffer B (1mM triethanolamine pH 7.4), and loaded onto a 1 ml MonoQ column (GE Healthcare), equilibrated in buffer C (20 mM triethanolamine pH 7.4, 1 mM EDTA, 50 mM NaF, 1 mM DTT, 1 mM benzamidine, 0.1 mM PMSF, 0.1% Triton X-100). The column was developed with 30 ml gradient of 0–500 mM NaCl in buffer C. Fractions were analyzed by SDS-PAGE, divided in small aliquots, snap-frozen in liquid N2 and stored at -80°C.
Cell culture, transfections and treatments
HEK-293T human embryonic kidney cells (ATCC, Manassas, VA, USA) were maintained in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented with 10% fetal calf serum (Gibco) and penicillin/streptomycin (100 U/ml; Gibco). For DNA transfection experiments, cells were seeded in 10 cm dishes, allowed to adhere overnight, and transiently transfected with constructs of interest using 1 μg DNA and 4 μl of the transfecting reagent Metafectene (Biontex, Germany) according to the manufacturer instructions. Cells were harvested 48 h after transfection. For RNA interference experiments, shEV (empty vector pRS), shKAP1 (OriGene Technology) or shMDM2 were transfected in HEK-293T cells to generate stable cell lines, as described.
Western blot, Far-Western blot and Immunoprecipitation
Mammalian cell proteins were extracted using ice-cold lysis buffer (50 mM Tris-HCl pH 7.5, 120 mM NaCl, 20 mM NaF, 1 mM EDTA, 6 mM EGTA, 15 mM Na-pyrophosphate, 0.5 mM Na-orthovanadate, 1 mM benzamidine, 0.1 mM PMSF and 1% Nonidet P-40). Protein concentration was determined using the Bio-Rad Protein Assay Reagent (Bio-Rad). Proteins were separated on SDS-polyacrylamide gels, transferred to polyvinylidene difluoride (PVDF) (GE-Healthcare), and probed with appropriate antibodies. Immune-complexes were revealed using the enhanced chemiluminescence (ECL) system (GE-Healthcare). Far-Western blotting was performed according to established protocols. Immunoprecipitation and immunoblot analysis were performed as described previously. To ensure that the observed interactions were not DNA-mediated, ethidium bromide was included in all samples during immunoprecipitation.
All reagents were prepared in MilliQ-ddH2O. Polyacrylamide gels were fixed in 50% methanol, 5% acetic acid in ddH2O for 20 min, washed with 50% methanol in ddH2O, then additionally for 10 min in ddH2O. The gel was soaked in 0.02% sodium thiosulfate for 1 min, washed twice with ddH2O for 1 min, soaked in cold 0.1% silver nitrate solution for 20 min at 4°C in the dark. The gel was then washed twice with ddH2O for 1 min and developed in 0.04% formaldehyde in 2% sodium carbonate with continuous, gentle shaking. Development was terminated by addition of a 5% acetic acid solution.
Silver stained protein bands were excised from the gel, reduced with 10 mM DTT, alkylated with 55 mM iodoacetamide and cleaved with porcine trypsin (Promega, Madison, USA) in 50 mM ammonium bicarbonate (pH 8.0) at 37°C overnight. The extracted peptides were analyzed by capillary liquid chromatography tandem mass spectrometry (LC-MS/MS) using a Magic C18 100 µm × 10 cm HPLC column (Swiss BioAnalytics, Switzerland) connected on line to a 4000 Q Trap (MDS Sciex, Concord, Ontario, Canada) as described earlier. Peptides were quantified with the software Progenesis LC (Nonlinear Dynamics, Durham, NC, USA).