FIGURE 2.

Phosphorylation of GlgE in mycobacteria. E. coli harboring pETPhos_glgE was used as a source of nonphosphorylated GlgE (GlgE), and the strain harboring pDuet_glgE coexpressing PknB and GlgE provided the phosphorylated GlgE isoform (GlgE-P). GlgE and GlgE_Ala were produced in M. bovis BCG strains harboring pVV16_glgE or pVV16_glgE_Ala, respectively. Three μg of purified His-tagged GlgE derivatives were analyzed by SDS-PAGE after staining with Coomassie Blue (upper panel), detected on independent SDS-PAGE gels by immunoblotting using anti-phosphothreonine (middle panel) or anti-phosphoserine (lower panel) antibodies according to the manufacturer's instructions (Invitrogen), and revealed with secondary antibodies labeled with IRDye infrared dyes (Odyssey Classic). M, molecular mass markers.