NleL Modulates Pedestal Formation Figure 3. E3 ligase activity of NleL is involved in modulating EHEC pedestal formation. HeLa cells were infected at a multiplicity of infection of 100 with wild type EHEC, nleLC753A, or nleLC753A harboring plasmid expressing wild-type NleL. Cells were infected for 6 hours. (A) Bacteria were visualized by staining with an anti-EHEC LPS antibody (green). Actin was detected with a Texas Red Phalloidin (red). (B) The number of micro- clusters of pedestals on HeLa cells were counted and were grouped into clusters having 1–4 pedestals, 5–10 pedestals and .10 pedestals. Quantitative analysis includes three independent experiments. A minimum of 300 cells were counted from each experiment with standard deviation shown as error bars. doi:10.1371/journal.pone.0019331.g003 ately digested cyaA reporter gene plasmid (pZP599) [62]. The HA- tagged EHEC Tir complementation was previously described [9]. The plasmid expressing amino acid residues 59 through 782 of NleL (NleL59–782) as a fusion protein with glutathione S-transferase (GST) was constructed by subcloning the relevant nleL coding sequence into pGEX-KG (pZP1173) [63]. Single base substitution mutations changing residues either C688 (pZP1667) or C753 (pZP1668) to Serine or Alanine were introduced using the Quick-Change Site- Directed Mutagenesis Kit (Stratagene, La Jolla, CA) according to the manufacturer’s protocol. The plasmid expressing C-terminal 6xHis- epitope tagged Tir (pZP1517) was constructed by cloning Tir coding sequence into pET28a (EMD Biosciences, Madison, WI). HeLa cells from ATCC Cell Biology Stock Center (Manassas, VA), were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (Gibco, Carlsbad, CA, USA). Purification of recombinant proteins from E. coli Recombinant fusion proteins, GST-NleL and Tir-6XHis were purified from E. coli BL21(DE3) harboring respective plasmids using glutathione- Sepharose 4B (Amersham Biosciences, Piscat- away, NJ) and Ni-NTA resin (Qiagen, Valencia, CA), respectively. All purified proteins were dialyzed and resuspended in phosphate- buffered saline (PBS) containing 2 mM dithiothreitol. PLoS ONE | www.plosone.org 5 April 2011 | Volume 6 | Issue 4 | e19331