Immunoprecipitation and CIAP treatment

Cells were rinsed with PBS and lysed with modified RIPA buffer (150 mM NaCl, 50 mMTris pH7.5, 1% NP40, 0.25% Sodium deoxycholate, 1mM EDTA, 1mM PMSF, 1mg/ml aprotinin, leupeptin, pepstatin, 1mM Na₃VO₄, 1mM NaF). 1 mg total cellular protein were incubated with 5 μl mouse anti-BCL10 antibody (sc-5273, Santa CrutzBiotchnology) for 16 hr at 4°C. 50 μl protein A beads were then added. The whole mixtures were incubated at 4°C for 4 hr. Protein A beads were spun down, washed twice with RIPA buffer and subjected to Calf Intestine Alkaline Phosphatase (CIAP) (New England Biolab.) treatment as suggested by the manufacturer’s protocol.