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. 2013 May 6;2:10.3402/jev.v2i0.20181. doi: 10.3402/jev.v2i0.20181

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© 2013 Erika Bartolini et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Fig. 2 — CM rHtrA expressed by E. coli BL21(DE3) ΔtolR(pET-htrA) is associated with the OMV fraction and is partially detected on the OMV surface. (A) Western blot analysis of CM rHtrA expression and compartmentalisation. Mid log cultures of E. coli BL21(DE3) ΔtolR(pET-htrA) (marked as ΔtolR(pET-htrA)) were induced with 1 mM IPTG for 3 h. CM rHtrA expression was assessed by Western blot in total bacterial extracts (TE), culture supernatant before (S1) and after (S2) OMV purification and in the purified OMV fraction. Each loaded sample corresponded to 5 mL of the original bacterial culture. OMVs purified from the recipient strain E. coli BL21(DE3) ΔtolR(pET) (empty OMV) and IMAC-purified CM rHtrA were run simultaneously as controls. As shown in the figure, CM rHtrA exists in 2 major protein species of approximately 48 and 20 kDa, representing the full length and a processed form of the protein, as verified by MALDI-TOF analysis. Both bands are also visible in the ΔtolR(pET-htrA)-derived samples. CM rHtrA bands show a slightly higher molecular weight due to the presence of the histidine tag. Molecular weight markers are on the left. (B) Estimation of CM rHtrA amount present in CM rHtrA-OMVs by comparative Western blot. Different quantities of CM rHtrA-OMVs and CM rHtrA were analyzed by immunoblot and the amount of rHtrA present on CM rHtrA-OMVs was estimated by densitometric analysis of the 48-kDa HtrA band (boxed). (C) FACS analysis of rHtrA surface exposure on CM rHtrA-OMV. Purified CM rHtrA-OMV (empty peak) and “empty” OMVs (shaded peak) were incubated with anti-CM rHtrA antibodies. CM rHtrA-OMV were also incubated with antibodies raised against E. coli contaminants (dotted peak) as an additional negative control. The binding was revealed using an Alexa Fluor 647 labelled secondary antibody. (D) Immuno electron microscopy analysis of CM rHtrA-OMV. CM rHtrA-OMVs and empty OMVs were incubated with anti-CM rHtrA antibodies and the presence of rHtrA was detected with a secondary gold-conjugated antibody. Arrows indicate HtrA immunogold staining. Bars represent the image size scale.