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. 2015 May 5;6(3):e00354-15. doi: 10.1128/mBio.00354-15

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Copyright © 2015 Michard et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

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FIG 1 — LegK2 interacts with ARPC1B and ARP3 subunits of the ARP2/3 complex. (A) Yeast two-hybrid assays of L. pneumophila protein kinase LegK1/LegK2 and ARPC1B/ARP3 subunits of the human ARP2/3 complex. Diploids from the mating of AH109(pGBKT7-legK1) or AH109(pGBKT7-legK2) with Y187(pACT2), Y187(pACT2-ARPC1B), or Y187(pACT2-ACTR3) were grown on SD medium without tryptophan (−W) or leucine (−L). Histidine auxotrophy was tested by plating 5 or 50 µl of diploids on SD-W-L medium without histidine (−H) in the presence of 10 mM 3-AT. (B) Affinity copurification of Flag-tagged ARP2/3 subunits with GST-tagged LegK2 protein. HEK293T cells were cotransfected with pDEST27, pDEST27-legK2, or pDEST27-legK2_K112M and pCI-Neo3Flag-ARPC1B or -ACTR3. GST, GST-tagged LegK2, or LegK2_K112M was purified on glutathione-agarose 4B, and purified fractions were immunoblotted with both anti-GST and anti-Flag antibodies. (C) Cellular localization of ARPC1B/ARP3 and LegK2/LegK1 proteins in HEK293T cells cotransfected with pDEST27, pDEST27-legK2, or -legK1 and pCI-Neo3Flag-ARPC1B or -ACTR3. The GST, GST-LegK2, and GST-LegK1 proteins were detected by immunofluorescence with anti-GST antibodies (green), and 3Flag-ARPC1B and 3Flag-ARP3 were detected with anti-Flag antibodies (red). Scale bars, 10 µm. (D) Quantitation of colocalization by Pearson coefficient. The Pearson coefficient was determined with the JACoP plugin of the ImageJ software and is expressed as the mean value calculated for 30 cells.