Figure 3.

Ubiquitin ligase activity of recombinase activating gene 1 (RAG1) mutants. (a) Sample auto-ubiquitylation assay performed as described in the Materials and methods using RAG1[218–389] wild type (WT) and CDC34 as indicated. Products were analysed by western blot with anti-Xpress antibody followed by anti-mouse-horseradish peroxidase (HRP) antibody. The positions of unmodified (R1), mono- and poly-ubiquitylated RAG1 are indicated. (b) Karyopherin alpha 1 (KPNA1) ubiquitylation assays were performed as described in the Materials and methods using the E2 enzyme UbcH2 or UbcH5a and RAG1[218–389] WT. The percentage of KPNA1 converted to mono- (Ubi₁), di- (Ubi₂), tri- (Ubi₃) and tetra-ubiquitylated (Ubi₄) species was determined by western blot for each sample. The average and standard deviation for three independent trials are shown. (c) Ubiquitin ligase assays were performed as described in (a) using the WT or various mutants of RAG1[218–389]. The percentage of RAG1 converted to mono- (Ubi₁), di- (Ubi₂), tri- (Ubi₃) and tetra-ubiquitylated (Ubi₄) species was determined by western blot for each sample. The average and standard deviation for at least three independent trials are shown; the significance of the decrease in auto-ubiquitylation relative to WT was determined by Student’s t-test (*P < 0·05; **P < 0·01). I/C/S, triple mutant I292A/C317A/S327F. (d) Partial proteolysis was performed with trypsin as described in the Materials and methods. Products were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) on an 18% gel and analysed by western Blot as described previously.⁶ Open arrows, undigested RAG1[218–389]; filled arrows, cleavage product spanning amino acids 254–377. Please note that extraneous lanes have been excised between the I/C/S and S327F samples and between H270A and N265A.