FIG 3.

Expression of mgtC_MAB in Sauton's medium and inside macrophages. (A) Quantification of mgtC_MAB (MAB_3593), MAB_3592c, and MAB_0146 RNA expression by qRT-PCR using RNA isolated from the wild-type strain grown in medium with a high or low Mg²⁺ concentration or internalized for 24 h inside J774 macrophages. The 16S rRNA gene was used as an internal standard. Results are expressed as means + standard deviations of the data from three experiments performed in triplicate. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student t test). (B) Expression of M. abscessus MgtC protein from wild-type M. abscessus grown under conditions of a high or low Mg²⁺ concentration or internalized for 1 or 24 h inside J774 macrophages. Lysate of M. abscessus mgtC mutant grown with or without Mg²⁺ was loaded as a negative control. Bacterial lysates equivalent to 4 μg (lysates from bacterial cultures) or 20 μg (lysates from infected macrophages) of total proteins were separated using 12.5% SDS-PAGE. For the detection of MgtC_MAB, the membrane was immunostained using a hyperimmune serum specific for MgtC_MAB (see Fig. S5 in the supplemental material), washed, and probed with peroxidase-conjugated goat anti-mouse (IgG; 1/5,000). As a control, the samples were immunostained with an immune serum specific for the KasA protein of M. tuberculosis.