Phosphorylation of ParB in mycobacteria.

<p><i>E</i>. <i>coli</i> harboring pETPhos_<i>parB</i> was used as a source of non phosphorylated ParB (ParB), and the strain harboring pDuet_<i>parB</i> coexpressing PknB and ParB provided the phosphorylated ParB isoform (ParB-P). ParB and ParB_Ala were produced in <i>M</i>. <i>smegmatis mc</i>^<i>2</i><i>155</i>Δ<i>parB</i> strains harboring pVV16_<i>parB</i> or pVV16_<i>parB</i>_<i>Ala</i>, respectively. Three μg of purified His-tagged ParB derivatives were migrated and 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, LiCOR). <i>M</i>, molecular mass markers.