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. Author manuscript; available in PMC: 2010 Nov 30.

Published in final edited form as: Mol Microbiol. 2008 Oct 24;70(6):1378–1396. doi: 10.1111/j.1365-2958.2008.06487.x

Translocation of TEM1∷VceA and TEM1∷VceC into J774 macrophages. Cytosolic translocation of β-lactamase by wild type (2308) or ΔvirB2 mutant strain (ADH3) of B. abortus was assessed by fluorescence microscopy. Cells in which translocation of the fusion protein has occurred appear blue. (A) Quantification of effector translocation. Cells from ten independent random microscope fields were counted and the percent blue cells calculated. Results shown are the mean ± SD of three independent experiments. (B) TopB. abortus 2308 containing the control fusion protein pFlagTEM1-GST, showing lack of translocation of FLAG-TEM1∷GST. Second from top to bottomB. abortus 2308 (left) or virB2 mutant (right) expressing FLAG-TEM-1 fused to B. abortus VceA or the C-terminal domain of VceC from B. abortus or B. suis. Results are from a representative individual experiment that was repeated three times independently. (C) Western blots showing equal expression levels of FLAG-TEM1 fusion proteins in B. abortus wild type and virB2 mutant. Proteins were detected using anti-FLAG antiserum (upper row). As a loading control, the blot was probed with anti-Bcsp31 (lower row).

Translocation of TEM1∷VceA and TEM1∷VceC into J774 macrophages. Cytosolic translocation of β-lactamase by wild type (2308) or ΔvirB2 mutant strain (ADH3) of B. abortus was assessed by fluorescence microscopy. Cells in which translocation of the fusion protein has occurred appear blue. (A) Quantification of effector translocation. Cells from ten independent random microscope fields were counted and the percent blue cells calculated. Results shown are the mean ± SD of three independent experiments. (B) TopB. abortus 2308 containing the control fusion protein pFlagTEM1-GST, showing lack of translocation of FLAG-TEM1∷GST. Second from top to bottomB. abortus 2308 (left) or virB2 mutant (right) expressing FLAG-TEM-1 fused to B. abortus VceA or the C-terminal domain of VceC from B. abortus or B. suis. Results are from a representative individual experiment that was repeated three times independently. (C) Western blots showing equal expression levels of FLAG-TEM1 fusion proteins in B. abortus wild type and virB2 mutant. Proteins were detected using anti-FLAG antiserum (upper row). As a loading control, the blot was probed with anti-Bcsp31 (lower row).