H. pylori and PaCS Figure 6. Colocalization in PaCS of bacterial products, UPS and oncogenic/signaling molecules in vivo and in vitro. (A, 20,000x; enlarged in a1, 95,000x, and a2, 120,000x) Colocalization in PaCSs of CagA (15 nm gold particles) with polyubiquitinated proteins (10 nm gold particles) and 20S-b5i subunit of immunoproteasome (5 nm gold particles). Note in a1 and a2 close reciprocal contacts of some 20-nm gold particles with 10- or 15-nm ones; also note in a2 the linear-punctate substructure of PaCS particles. (B, 80,000x) In another section through the same PaCS as of A, colocalization of CagA (15 nm gold) and SHP2 protein (10 nm gold) is obtained. (C, 5,000x; enlarged in c, 35,000x) Another PaCS showing selective ERK reactivity. (D, 10,000x; enlarged in d, 45,000x) In addition to a typical particulate PaCS (left part of d) immunoreactive for FK1 antibodies (recognizing polyubiquitinated proteins), another, FK1-unreactive non-particulate cytoplasmic structure (upper right of D and d) with a filamentous- honeycomb meshwork. (E, 6,300x) Dysplastic cell with prominent nucleolus showing several PaCSs (one labelled with asterisk) in its basal cytoplasm. A PaCS containing bacterial remnants is enlarged in e (20,000x) to show H. pylori (OMPs) immunoreactivity of both bacterial body and PaCS itself. n, nucleous; nl, nucleolus. (F) Confocal microscopy of HeLa cells (nuclei in blue) incubated for 24 h with H. pylori BCF shows the presence in the cytoplasm of yellow spots (arrows; see also enlargement in f) representing the colocalization of ubiquitinated proteins (FK2; green) with VacA (red). Note that vacuole-associated VacA (arrowheads) does not colocalize with FK2. Colocalization charts (graphs 1 and 2) show the intensity profile for each fluorescence taken along the dotted lines. Pictures are from one single confocal section. Bar: 25 mm. doi:10.1371/journal.pone.0009716.g006 PLoS ONE | www.plosone.org 8 March 2010 | Volume 5 | Issue 3 | e9716