Figure 5

Localization of α7nAChRs in cultured cardiomyocytes. (A) Immunoblot analysis of α7nAChR (55 kDa) in total lysates of neonatal rat ventricular myocytes (NRVM) and adult human cardiomyocytes AC16. (B) FACS analysis of intact AC16 cells sorted by intensity of bound anti-α7nAChR antibody versus forward scatter (FSC). Contour map (left) shows distinct populations of cells that are quantified in the histogram (right): cells in peak-b are bound with fluorescence-labeled antibody; cells in peak-a were incubated with antibody preadsorbed with peptide immunogen; cells incubated with isotype control IgG are in peak-c. (C) Fluorescence microscopy localized α7nAChR (a7nR) in NRVM using a rabbit anti-α7nAChR antibody and FITC-labeled secondary antibody. Counterstaining with TRITC-labeled phalloidin identified F-actin in cardiomyocytes. Using identical exposure settings, isotype rabbit IgG showed low background fluorescence in myocytes stained for F-actin. Scale bars represent 20 µm. (D) Micrographs taken at the same exposure settings show strong localization of anti-α7nAChR antibodies to NRVMs while the IgG isotype control is weakly fluorescent. Scale bars, 20 µm.