Figure 3: Unchanged LEMD2 Localization and Expression in Patient Cells, Cardiac Tissue, and Transfected C2C12 Cells

(A and B) Representative confocal images of patient and control myocardial tissue as well as fibroblasts showing normal localization of LEMD2 (green, arrow) co-localizing with lamin A/C (red) at the nuclear membrane; 4′,6′-diamidino-2-phenylindole (DAPI) (blue) indicates nuclei. Scale bars represent 20 μm. (C) Western blot showing LEMD2 and lamin A/C protein expression in fibroblasts in the left panel. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control. Quantification by densitometry (n = 3 experiments with 2 to 3 replicates) of LEMD2; quantification of lamin A and C and LEMD2 protein expression in the right panel shows no difference. (D) Representative confocal images of transfected C2C12 cells with recombinant enhanced green fluorescent protein (eGFP)-tagged wild-type (WT) (p.L13) or mutant (p.R13) LEMD2 (green) co-transfected with mCherry–lamin A/C (LMNA) (red) demonstrated co-localization. Scale bar represents 20 μm. (E) Western blot analysis of eGFP-LEMD2 proteins after transfection into HEK293 cells. GAPDH was used for loading control. Quantification by densitometry (n = 3) reveals no difference in the protein level between mutant (p.R13) and WT (p.L13) LEMD2. Un = un-transfected; other abbreviations as in Figure 2.