Figure 7.

Coimmunoprecipitation, DNA cleavage activity ex vivo, and RSS binding activity in vivo, of RAG proteins expressed in mammalian cells. (A) Full-length untagged RAG1 protein (wild-type or mutants) and polyhistidine/myc tagged full-length RAG2 proteins were coexpressed in 293T cells. The lysate was subjected to immunoprecipitation using anti-myc antibodies. Five microliters of lysate (input, lanes 1–4) and half of the precipitated proteins (IP, lanes 5–8) were analyzed on a 7.5% SDS–polyacrylamide gel, and after transfer to a membrane, proteins were detected with anti-RAG1 (R1P1) and anti-RAG2 antibodies. Asterisk indicates breakdown products of the RAG1 proteins. (B) GST-core RAG1 and full-length untagged RAG2 were coexpressed in 293T cells and purified by glutathione affinity chromatography. Yields of RAG1 and RAG2 were equal for wild-type and mutant RAG1 proteins (not shown). Cleavage activity was assessed in a standard coupled cleavage reaction using serial twofold dilutions of the partially purified RAG proteins. (C) One-hybrid in vivo DNA binding assay. The ability of the mutant RAG1 proteins to interact with RAG2 and the 12-RSS was determined using a mammalian one-hybrid system. Cells were transfected with the p(12)₈ reporter plasmid (expressing firefly luciferase), pRL-CMV (expressing renilla luciferase), and plasmids expressing GST-core RAG1 and core RAG2-VP16. Firefly luciferase values were normalized by dividing them by the renilla luciferase values, which corrects for variations in transfection efficiency. The normalized value obtained in transfections of RAG2 alone was arbitrarily set to one, and all other values are expressed relative to this. The values represent the mean of results from three independent transfections, and error bars indicate the standard error of the mean.