Supplemental Figures

This article contains the following supporting material:

• Supplemental Figures 1-7.pdf - Supplementary Figure 1. α-Abl and α-Arg antibodies are specific.
  a-b. Images of Abl^-/-/Arg^-/- cells infected with EPEC and stained with DAPI, FITC-phalloidin, and α-Abl mAb 8E9-Cy3. In these merged images, bacteria are pseudocolored blue, Abl red, and actin green. Note that α-Abl mAb 8E9 does not detect other proteins in cells lacking Abl and Arg.
  c-d. Images of Abl^-/-/Arg^-/- cells transfected with Arg-YFP, infected with EPEC and stained with DAPI, and α-Arg pAb-Cy5. In these merged images, bacteria are pseudocolored blue, Arg-YFP green, and α-Arg red. Note that α-Arg pAb does detect Arg-YFP (cell on left), but no other proteins in cells lacking Abl and Arg (cell on right - arrow).
  e-f. Images of Abl^-/-/Arg^-/- cells transfected with HA-Abl, infected with EPEC and stained with DAPI, α-HA 3F10-FITC, α-Arg pAb-Cy3. In these merged images, bacteria are pseudocolored blue, Arg red, and HA-Abl green. Note that α-Arg pAb does not detect overexpressed HA-Abl.
  g-h. Images of Abl^-/-/Arg^-/- cells transfected with Arg-YFP, infected with EPEC and stained with DAPI and α-Abl mAb 8E9-Cy5. In these merged images, bacteria are pseudocolored blue, Arg-YFP green, and Abl red. Note that α-Abl mAb 8E9 does not detect overexpressed Arg-YFP. Scale bars represent 5 μm.
  
  Supplementary Figure 2. N-Src localizes in pedestals but c-Src does not.
  a-d. Images of 3T3 cells infected with EPEC. Cells were stained with DAPI (a), FITC-phalloidin (b), and α-Src pAb (c). In the merged image (d), bacteria are pseudocolored blue, Src red and actin green. Note that endogenous Src does not localize in pedestals.
  e-h. Images of 3T3 cells expressing exogenous c-Src-WT and infected with EPEC. Cells were stained with DAPI (e), FITC-phalloidin (f), and α-Src pAb (g). In the merged image (h), bacteria are pseudocolored blue, Src red and actin green.
  i-l. Images of 3T3 cells expressing green fluorescent protein (GFP(k)) and infected with EPEC. Cells were stained with DAPI (i), and Alexa 594-phalloidin (j). In the merged image (l), EPEC are pseudocolored blue, actin red, and GFP green.
  m-p. Images of 3T3 cells transfected with N-Src-WT, infected with EPEC and stained with DAPI (m), FITC phalloidin (n), and α-Src pAb (o). In the merged image (p), bacteria are pseudocolored blue, actin green, and N-Src red. Note that N-Src is localized in the pedestal. Scale bars represent 5 μm.
  
  Supplementary Figure 3.
  a-d. EPEC forms pedestals on cells lacking Src, Fyn and Yes. Image of Src^-/-/Fyn^-/-/Yes^-/- cells stained with DAPI (a) and FITC-phalloidin (b) and α-phosphotyrosine (c). In the merged image (d), bacteria are pseudocolored blue, actin green, and phosphotyrosine red. Note that pedestals form on these cells.
  e-j. Src does not localize in pedestals on Abl^-/-/Arg^-/- cells. Images of Abl^-/-/Arg^-/- cells infected with EPEC and stained with DAPI (e), α- Src pAb (f), FITC-phalloidin (h) and α-phosphotyrosine (i). In the merged images (g,j), bacteria are pseudocolored blue, actin green, and src (g) or phosphotyrosine (j) red. Scale bars represent, 5 μm.
  
  Supplementary Figure 4. Structure of PD166326 and SKI-DV-1-10 (DRV-1)
  
  Supplementary Figure 5. PD166326 does not block motility of Listeria monocytogenes or Shigella flexneri.
  a-b. Images of 3T3 cells treated with DMSO (a,c) or PD (b,d), infected for 18 hrs with Listeria (a,b) or for 6 hrs with Shigella, and stained with DAPI to recognize bacteria, and with FITC-phalloidin to recognize actin. The presence of actin surrounding entering bacteria and actin comet tails inside and protruding from cells indicates that PD does not nonspecifically affect actin polymerization. Scale bar, 5 μm.
  
  Supplementary Figure 6. Expression of PD-resistant mutant Abl-T315I blocks loss of tyrosine phosphorylation when PD is added after pedestals have formed.
  a-l. Images of 3T3 cells transfected with vector (a-d), Abl-WT (e-h), or Abl-T315I (i-l), infected with EPEC for 5 hrs, and treated with PD for 15 min. Cells were stained with DAPI to recognize EPEC, FITC-phalloidin to recognize actin, α-Abl at low concentration to only detect transfected Abl, and α-phosphotyrosine mAb 4G10. Note that transfected Abl (g) and Abl-T315I (k) localize in the pedestal, and that pedestals are still evident at this time point. Note also loss of P-Tyr staining in cells transfected with vector (d) or Abl-WT (h), but not with Abl-T315I (l). Scale bars, 5 μm.
  
  Supplementary Figure 7. Expression of PD-resistant mutants of Src and N-Src fail to prevent loss of tyrosine phosphorylation when PD is added after pedestals have formed.
  a. PD-166326 fails to block kinase activity of v-Src-338I. Analysis of kinase activity in 293 cells transiently transfected with v-Src-338I. Transfected v-Src-338I was immunoprecipitated with α-Src pAb and incubated with GFP-IYGEF and ATP, together with DMSO (lanes 1) or 10 nM PD-166326 (lane 2), 100 nM PD-166326 (lane 3), or 1 μM PD 166326 (lane 4). Phosphotyrosine was detected by western analysis with α-4G10 (upper panel). The blot was reprobed with α-Src to insure equal loading of protein (lower panel). Kinase activity from endogenous Src was not detectable in these cells (not shown), 1샽 PD blocked kinase activity of v-Src-338I by only 50%. The Ki of PD for c-Src is 10 nM (Kraker et al., 2000); and not shown).
  b-i. Images of 293 cells transfected with v-Src-338I (b-e) or N-Src-T346M (f-i), infected with EPEC for 5 hrs, and treated with PD for 15 min. Cells were stained with DAPI to recognize EPEC (b,f), FITC-phalloidin to recognize actin (c,g), α-Src pAb to recognize transfected v-Src-338I (d) or N-Src-T346M (h), and α-phosphotyrosine mAb 4G10 (e,i). Note that pedestals are still evident at this time point, but p-Tyr staining was lost in all cells including those transfected with v-Src-338I (e) or N-Src-T346M (i). Scale bars, 5 μm.
  

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