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Blood, Vol. 107, Issue 1, 373-380, January 1, 2006
CD163 is the macrophage scavenger receptor for native and chemically modified hemoglobins in the absence of haptoglobin
Blood Schaer et al.
107: 373
Supplemental materials for: Schaer et al
Files in this Data Supplement:
- Figure S1. Exclusion of endogenous Hp synthesis by HEK293 cells and exclusion of Hp contamination of the purified Hb preparations (JPG, 29.1 KB)
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A. Hp Western blot was performed with either lysate or cell culture supernatant from HEK 293 cells. For the analysis of cell lysates, confluent layers of different passages of HEK293 were lysed with 200 µl of cell lysis reagent per well (6-well plate; CelLytic-M, Sigma). After 1:1 dilution with sample buffer (reducing conditions), 50 µl samples were run on a 10% SDS PAGE gel (Criterion precast gels, Biorad). After blotting on PVDF, Hp was detected with a polyclonal rabbit antibody specific to human Hp (Dako) and a HRP goat anti-rabbit secondary antibody (Amersham), respectively. Immunoblots were developed using ECL chemilumi-nesence reagent (Amersham). Chemiluminescence signals were acquired with a Chemi-Doc XRS instrument and Quantity One software (Biorad). Different concentrations of purified human Hp (Sigma) were used as positive controls. B. The different Hb preparations used in these studies were analyzed for potential Hp contamination on an Agilent 2100 Bioanalyzer according to the manufacturer’s instruction. Purified Hp (Sigma) was used as a positive control andcould be detected at dilutions up to 1:100 (Hp:Hb). No Hp  -chain peak was detectable in the analyzed Hb preparations.
- Figure S2. Free Hb competitively inhibits Hb-Hp uptake in human macrophages (JPG, 25.6 KB)
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Excess unlabeled Hb and Hb–Hp (but not free Hp) inhibit the uptake of the fluorescent indicator ligand (fl). Human dexamethasone-treated monocytes, which have a high functional CD163 expression, were incubated with fluorescent ligand and a 100-fold excess of unlabeled competitor for 30 min. Identical experiments were performed in which either Hb or Hp, as part of the Hb–Hp complex, was coupled to Alexa-488. The bars represent mean ± SD of uptake assays from one representative experiment performed in triplicate.
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