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. 2017 Jan 23;8:14188. doi: 10.1038/ncomms14188

Figure 5. Quaternary structure of CRP in inflamed tissue.

Figure 5

(a) Tissue lysates of burn wounds and healthy skin were separated on denaturing SDS–PAGE, which induces dissociation of pCRP. Anti-CRP-8 antibodies detected a band in burn wound lysates, which had the size of a CRP monomer (23 kDa). (b) Immunohistochemistry of burn wounds revealed distinct staining (green) by anti-mCRP-9C9 antibodies, but only minimal staining with anti-pCRP-8D8 antibodies. Scale bars, 100 μm. (c) The oxidation state of CRP in burn wounds was assessed by non-reducing SDS–PAGE. Reduced CRP migrates slower and can thus be distinguished from oxidized CRP. We did not observe reduced CRP in tissue lysates of burn wounds. (d) The quaternary structure of CRP in human skeletal muscle after I/R injury (two patients, total of six samples), burn wounds (three patients, total of five samples) and CEA specimens (one patient, total of two samples) was determined by assessing the accessibility of the intrasubunit disulfide bond. Tissue lysates were incubated with 10 mM DTT at 37 °C to assess the accessibility of the intrasubunit disulfide bond. DTT can efficiently reduce the disulfide bond in mCRP, but not in pCRP/pCRP*. Small amounts of tissue-deposited CRP could be reduced by DTT (indicated by an asterisk). Reduction was effective if lysates were heated, which induces pCRP dissociation. Quantification of reduced CRP revealed a significant difference between control pCRP and CRP in tissue lysates of burn wounds and CEA plaques. Displayed are means and s.e.m. P values were calculated with an unpaired t-test. *P value <0.05, **P value <0.01 and ***P value <0.001. (e) Tissue lysates were separated on 1/20 SDS–PAGE. Anti-CRP-8 antibodies recognized a distinct band in burn wound lysates that migrated at the same height as control pCRP. It did not recognize a band with similar size to mCRP. A band, which could be observed in burn wound lysates and healthy skin, was also recognized (#) and most likely reflects binding to a common skin epitope. (f) Accessibility of the intrasubunit disulfide bond in CRP in the presence of PC/LPC liposomes and microvesicles (MV). Control mCRP was effectively reduced. No dissociation of pCRP to mCRP was observed. (g) Lipid binding does not affect reduction of mCRP by DTT, as reduction of control mCRP incubated with liposomes was unimpeded. Uncropped images of western blots are shown in Supplementary Fig. 13.