The monoclonal antibody specificity tested by dot-blot analysis. (A) Summary of peptide sequences. Three TLS peptides containing either no modification (Non) or R216/R218me2a (me2a) or R216/R218me2s (me2s) were synthesized. TLS peptide containing no modification was used for producing polyclonal antibody in rabbit, and TLS peptides containing R216/R218me2a or R216/R218me2s were used for the immunization of mice and hydridoma development. (B and C) Antibody specificity was tested by dot-blot analysis. Diluted peptides (B; 0.2, 1, 5 ng, C; 20, 100, 500 ng) were blotted onto the nitrocellulose membrane and the dot-blotted membranes were incubated with a rabbit polyclonal antibody A1 (B) or a mouse monoclonal antibody 2B12 (C). Note that A1 reacted equally with TLS peptides either no modification or symmetrical or asymmetrical dimethylation, and 2B12 recognized only TLS peptide containing asymmetrical dimethylated arginines.