Detection of in vivo methylation of TLS. (A) Endogenous TLS is methylated in HeLa cells. Cell extracts from HeLa treated or not with 3 μM of the general methylation inhibitor AdOx for 24 h were used for immunoprecipitations. The extracts were immunoprecipitated with rabbit normal IgG or rabbit polyclonal anti-TLS antibody. The immunoprecipitated TLS were analyzed by western blotting with 2B12 or mouse monoclonal anti-TLS antibody. The input lane shows 10% of the protein used in each immunoprecipitation. Note that TLS methylation was inhibited by AdOx, and 2B12 specifically recognized methylated TLS. (B) Immunoprecipitation of endogenous methylated TLS from HeLa cell extracts was performed with 2B12 in the presence or absence of competing peptides used for immunization. Bound methylated TLS was eluted with SDS sample buffer resolved by SDS-PAGE, and analyzed by western blotting with rabbit polyclonal anti-TLS antibody.