Mouse monoclonal 4c5 anti ddk antibody

CTF135 cells (4.4 × 10⁶) cells were seeded in 150 mm dishes, cultured for 24 h, exposed to test compound or vehicle for 2 h and then heat-treated as described above. Cells were extracted on ice with lysis buffer (50 mM HEPES-KOH, pH 8.0, 100 mM KCl, 2 mM EDTA, 0.1% NP40, 10% glycerol) supplemented with 1 mM DTT, 1 mM PMSF, 0.25 mM sodium orthovanadate, 50 mM β-glycerolphosphate, 10 mM NaF, 5 nM okadaic acid, 5 nM calyculin A and protease inhibitor cocktail (Complete). Cells were lysed by three cycles of rapid freezing (dry ice-ethanol bath) and thawing (37°C water bath). Cell debris was then removed from cell lysates by centrifugation at 16 000 g for 20 min at 4°C. Protein concentrations of extracts were determined using the protein assay reagent of Bio-Rad and were equalized prior to further analysis. Aliquots of extracts were incubated with 50 μl magnetic beads conjugated with 4C5 anti-DDK mouse monoclonal antibody (OriGene Technologies) for 2 h at 4°C with gentle agitation. Immune complexes were collected by centrifugation and washed once with lysis buffer and then twice with rinsing buffer (20 mM Tris–HCl, pH 8.0, 2 mM CaCl₂). Immunoprecipitated proteins and aliquots of protein extracts were analyzed by immunoblotting using mouse anti-FLAG monoclonal antibody M5 (Sigma-Aldrich) or rabbit anti-human ATF1 monoclonal antibody (Abcam).