FIGURE 2.

RmlA is mainly phosphorylated by PknB with T12 site. (A) Phosphorylation of RmlA and mutants in M. smegmatis. The WT and mutants of rmlA genes were cloned into pMV261 with His tag and incorporated into M. smegmatis. The proteins were purified with Ni²⁺ agarose beads. With Western blotting, phosphorylated signals were detected with the antithreonine phosphorylation antibody. The experiment was performed in triplicates (n = 3). Error bars indicate standard error of mean. Asterisk indicates significant difference (P < 0.05). (B) Phosphorylation of RmlA with the pknB-KD in M. smegmatis. The RmlA protein was purified from strains with sgRNA 1 and sgRNA 3 targeting, which have strong repression of PknB expression, using anti-RmlA antibody, and pull-down with protein G beads. Through Western blotting, phosphorylated signals were detected with the antithreonine phosphorylation antibody. The experiment was performed in triplicates (n = 3). Error bars indicate standard error of mean. Asterisk indicates significant difference (P < 0.05).