Research Article

Clearance of Apoptotic Cells by Macrophages Induces Regulatory Phenotype and Involves Stimulation of CD36 and Platelet-Activating Factor Receptor

Figure 2

Colocalisation of PAFR and CD36 occurs during efferocytosis. BMDM were treated with PAFR agonist (PAF, 10−7 M) or apoptotic thymocytes (10 per macrophage) for 20 min to assess coimmunoprecipitation and colocalisation of PAFR and CD36. After washing, cells were lysed and subjected to immunoprecipitation and immunoblotting as described in Section 2, using antibodies to CD36 and PAFR (a). Another group was subjected to fixation prior to staining with anti-PAFR and anti-CD36 primary antibodies, followed by FITC- and PE-labelled secondary antibodies, respectively, and visualised by confocal microscopy as described in Section 2 (b). Quantification of colocalisation (c) was performed using Pearson’s coefficient and JACoP/ImageJ software, and data are presented as mean ± SEM of 15 pictures from three independent experiments ( versus vehicle). Protein expression was quantified by the AlphaEaseFC software v3.2 beta (Alpha Innotech). The autoradiographs show one representative experiment, and graph data are presented as mean ± SEM of three experiments ( versus control).