Fig. 7: Reduced N2Bus phosphorylation in FHL-1-deficient cardiomyocytes

a Total titin phosphorylation measured by ProQ Diamond/Sypro Ruby staining (left) and site-specific titin phosphorylation detected by western blot using antibodies to P-S3991 (N2Bus; middle) or P-S2080 (Z/I junction; right). Site-specific titin phosphorylation levels were normalized to total titin levels detected by WB using sequence-specific antibodies (Pan). Means were indexed to those of control (WT) groups. Data are mean ± s.e.m., n = 3 hearts/group, samples analyzed in triplicate; *p < 0.05, by two-tailed Student’s t-test. b, c, d Localization of PP5 (b), phospho-N2Bus^P-S3991 (c), and phospho-Z/I-junction^P-S2080 (d) in cardiomyocytes from FHL-1 WT and KO hearts by indirect immunofluorescence. Anti-PP5, anti-N2Bus^P-S3991 or anti-Z/I-junction^P-S2080 (secondary antibody: Cy3-conjugated IgG), counterstained with anti-PEVK antibody (secondary antibody: FITC-conjugated IgG). Bars, 5 µm (main) and 1 µm (insets). e, f Sarcomeric localization of phospho-N2Bus^P-S3991 (e) and phospho-Z/I-junction^P-S2080 (f) in FHL-1 WT and KO hearts by immunogold electron microscopy. Bars, 500 nm (main) and 100 nm (insets). Bar graph in e and f  shows average number of gold particles counted in 50-µm²-sized regions-of-interest (ROI), either on the sarcomeric I-band or elsewhere in the cardiomyocyte (‘Not on I-band’). Data in e and f are mean ± s.e.m., n = 5 ROIs from two hearts/group. In a and e, *p < 0.05 and ***p < 0.001, by two-tailed Student’s t-test