(A) Pneumocystis carinii (Pc) major surface glycoprotein (Msg) can significantly reduce Dectin-1-immunoreceptor tyrosine-based activating motif phosphorylation ((ITAM-p) by fungal β-glucans. A total of 2 × 106/well RAW 264.7 cells were preincubated with Msg as noted above, followed by fungal β-glucan induction for 1 hour. Determination of Dectin-1-ITAM-p state was noted as above. Representative blot of at least 3 separate experiments are shown. (B) The Dectin-1-ITAM signals were quantified with Image Studio Lite software and normalized to total Dectin-1 levels. (C) Enzyme-linked immunosorbent assay analyzing binding of Dectin-1 carbohydrate recognition domain (CRD) to Msg or Pc glucan as described under Materials and Methods. Wells were coated with 2 µ g/well of Pc Msg or Pc β-glucans overnight. After blocking, the respective hFc fusions were added and evaluated in duplicate wells. Although the hFc fusion control displayed no binding to either ligand, and hFc-Dectin-1 CRD did not demonstrate binding to Msg, significant binding of hFc-Dectin-1 CRD to Pc β-glucans was observed. Results represent the mean of 2 separate experiments. *, P < .05 and **, P < .01. BSA, bovine serum albumin; Sc, Saccharomyces cerevisiae.
