FIG. 3.
Anti-phospho-A20 S381 antibody recognizes A20 which is phosphorylated by IKKβ. (A) A phospho-specific antibody directed against A20 phospho-S381 was generated. To show specificity, Flag-A20 was cotransfected into HEK293T cells with GST-IKKβ and immunoprecipitated using an anti-Flag antibody. Immunoprecipitates were immunoblotted using the phospho-specific antibody for A20 S381. (B) Flag-A20 was cotransfected with GST-IKKβ into HEK293T cells. Lysates were subjected to immunoprecipitation using an anti-Flag antibody. Immunoprecipitates were treated with calf intestinal phosphatase (CIP) for 45 min at 37°C and then immunoblotted using the anti-phospho-A20 S381 antibody. The phospho-A20 S381 antibody no longer recognized calf intestinal phosphatase-treated A20. (C) Flag-A20 was cotransfected into HEK293T cells with GST-IKKβ. Cells were pretreated with 10 μM BMS-345541 or vehicle control for 3 h prior to lysis in order to facilitate dephosphorylation of A20 in the absence of IKKβ activity. Anti-Flag immunoprecipitates were washed four times with lysis buffer to remove BMS-345541 and three times with kinase buffer. Immunoprecipitates were then incubated a second time with 3 μM BMS-345541 or vehicle control for 20 min at room temperature in order to inhibit coprecipitating IKKβ activity, and an in vitro kinase assay was performed. Immunoblot analysis using the phospho-A20 S381 antibody showed in vitro phosphorylation of A20 S381 by coprecipitating IKKβ. (D) NEMO−/− or isogenic NEMO+/+ cells were transduced with pBABE-A20 retrovirus. Cells were selected in puromycin, and clones (>1,000) were pooled. Cells were treated with TNF-α (5 ng/ml) for the indicated times. Cells were then lysed, and A20 was immunoprecipitated by virtue of its Flag tag. Western blotting showed that in NEMO+/+ cells, A20 was phosphorylated in a TNF-α-dependent manner, while in NEMO−/− cells, A20 showed very little TNF-α-dependent phosphorylation.