Monoclonal Antibodies Specific for Disease-Associated Point-Mutants: Lamin A/C R453W and R482W
Figure 2
R453W antibody is specific for the EDMD associated lamin A/C R453W point-mutant.
(A) HeLa, HeLa Flag-Lamin A wt, HeLa Flag-Lamin A R453W, and HeLa Flag-Lamin A R482W cell lysates were separated by 7.5% SDS-PAGE and analyzed by Western blotting with the mouse monoclonal antibodies anti R453W (panel I), anti Flag M2 (panel II), anti Lamin A/C 4C11 (panel III), and β-actin (panel IV). (B) Peptide competition of R453W antibody. Binding of R453W antibody to Flag-Lamin A R453W was competed with increasing amounts (0.44, 2.2, 11, and 55pmol) of mutant “R453W Peptide” (NH2-449GKFVWLRNK457-COOH) or wild-type “wt Peptide” (NH2-449GKFVRLRNK457-COOH).Competition efficiency was examined by 7.5% SDS-PAGE/Western blot analysis of HeLa Flag-Lamin A R453W cell lysates. Control antibodies were: anti R453W not subjected to competition, anti Flag M2, anti Lamin A/C 4C11. (C) Immunofluorescence of a mixed population of HeLa and HeLa Flag-Lamin A R453W cells, seeded at a ratio of 3∶1. Cell staining was performed with anti R453W (top panel), anti Flag M2 (middle panel), and anti Lamin A/C 4C11 (bottom panel). DNA was counterstained with Hoechst 33342. Bar scale, 20 µm.