This is useful for detecting posttranslational modifications such as phos-phorylation. Phosphorylation can be measured by immunoblotting the immunoprecipitate using antiphosphorylated serine, threonine, or tyrosin antibody or autoradiography when TonEBP is immunoprecipitated from cells labeled metabolically with [32P]phosphate (Dahl et al., 2001; Lee et al., 2003).
Cells grown on a 100-mm dish are washed with ice-cold PBS and lysed with 0.1 ml of SDS extraction buffer (1% SDS, 50 mM Tris, pH 7.4, 5 mM EDTA, 2 mM PMSF, 20 mg/ml aprotinin, 12.5 mg/ml pepstatin, and 12.5 mg/ml leupeptin) at 4°. The lysate is boiled for 10 min and mixed with 0.9 ml Triton X-100 extraction buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Trition X-100, 2 mM PMSF, 20 mg/ml aprotinin, 12.5 mg/ml pepstatin, and 12.5 mg/ml leupeptin). The mixture is passed 10 times through a 25-gauge needle attached to a 1-ml syringe and then incubated on ice for 10 min. Protein concentrations of lysate are determined using a BCA-based assay kit (Pierce). For immunoprecipitation, 2mg of protein is incubated overnight at 4° with 10 ml anti-TonEBP serum followed by incubation with 30 ml of 50% protein A-Sepharose 4B beads (Sigma) for 4 h at 4° with constant agitation. Immune complexes are centrifuged for 1 min at 3000 rpm and are then washed three times in Triton X-100 extraction buffer. Immune complexes are treated with 30 ml 2 x SDS sample buffer after washing.
Was this article helpful?