Monoclonal Antibodies Specific for Disease-Associated Point-Mutants: Lamin A/C R453W and R482W
Figure 3
R482W antibody is specific for the FPLD-associated lamin A/C R482W point-mutant.
(A) 7.5% SDS-PAGE of HeLa, HeLa Flag-Lamin A wt, HeLa Flag-Lamin A R453W, and HeLa Flag-Lamin A R482W cell lysates followed by Western blot analysis with the mouse monoclonal antibodies anti R482W (panel I), anti Flag M2 (panel II), anti Lamin A/C 4C11 (panel III), and β-actin (panel IV). (B) Ig-fold competition of R482W antibody. Binding of anti R482W to Flag-Lamin A R482W was competed with recombinant HBcAg protein containing either the wild-type Ig-fold, “Ig-wt”, or the mutant Ig-fold, “Ig-R482W”, at increasing molar ratios. Competition efficiency was examined by 7.5% SDS-PAGE/Western blot analysis of HeLa Flag-Lamin A R482W cell lysates. Control antibodies were: anti R482W 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 R482W cells, seeded at a ratio of 3∶1. Cells were stained with anti R482W (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.