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. 2004 Oct;24(20):9207–9220. doi: 10.1128/MCB.24.20.9207-9220.2004

FIG. 4.

FIG. 4.

Artemis interacts with checkpoint proteins and is phosphorylated by ATM and ATR. (A, top blot) Artemis antibodies coimmunoprecipitate ATM, BRCA1, Rad50, Nbs1, and Mre11 from HeLa extracts. Protein A-Sepharose beads were used as a control. IP, immunoprecipitation; IgG, nonspecific immune serum. Immunoblotting (IB) was performed using antibodies to the indicated proteins. (A, lower blot) Reciprocal coimmunoprecipitation of Artemis by ATM antibodies. ATM+I, incubation of the extracts with DNase I prior to coimmunoprecipitation. (B) Depletion of Artemis in HeLa cells by transfection of siRNA eliminates the coimmunoprecipitation of Rad50 by Artemis antibodies. α-Artemis IP, immunoprecipitation with anti-Artemis antibodies; Pre., preimmune serum; Imm., Artemis antiserum. (C) Immunoprecipitation (IP) kinase assay shows that Artemis was phosphorylated by both ATM and ATR but by kinase-dead (kd) variants of the enzymes. The top shows an autoradiogram of Artemis phosphorylation by ATM or ATR. The bottom shows immunoblots of immunoprecipitated kinases used in the assays.