Fig. 1.

PtpA is present both in the cytoplasm and nucleus of host cells. a Confocal microscopy of U937 cells infected with WT BCG, or BCG ΔPtpA, or BCG (ΔPtpA + PtpA), or BCG (ΔPtpA + D126A) strain. Cells were infected at a multiplicity of infection (MOI) of 10 for 6 h. PtpA and PtpB were detected using primary antibody specific for PtpA or PtpB followed by DyLight 488-conjugated goat anti-rabbit IgG secondary antibody (green). Bacteria were stained with pHrodo Red succinimidyl (NHS) ester (Red). Nuclei were stained with DNA-binding dye (DAPI) (blue). Scale bars, 10 µm. Right, the percentage of cells with nuclear localization of PtpA or PtpB (a total of 200 cells were counted) and the survival of BCG strains in U937 cells infected for 6 h. b Time course analysis for intracellular localization of PtpA at early time points post-infection (2–6 h). Cells were stained as in a. Right, the percentage of cells with nuclear localization of PtpA and the survival of BCG strains in U937 cells infected for 2–6 h. c Immunoblot analysis of PtpA in U937 cells stablely expressing Myc-tagged Mtb PtpA. Cells were collected for fractionation to obtain the cytosolic fraction and the pellets containing the nuclei for immunoblot analysis. α-Tubulin and PARP were used as markers for the cytosolic and nuclear fractions, respectively. d Immunoblot analysis of PtpA and PtpB in U937 cells infected as in a. Cells were collected for fractionation to obtain the cytosolic fraction and the pellets containing the nuclei for immunoblot analysis as in c. *P < 0.05 and **P < 0.01 (unpaired two-tailed Student’s t-test). Data are representative of one experiment with at least three independent biological replicates (a, b; mean and s.e.m., n = 3). Full blots are shown in Supplementary Fig. 10