Monoclonal Antibodies Specific for Disease-Associated Point-Mutants: Lamin A/C R453W and R482W
Figure 5
R453W and R482W antibodies recognize the respective disease causing mutant A-type lamins in primary patient fibroblasts.
(A) Western blot analysis of wild type, EDMD (R453W), and FPLD (R482W) primary human fibroblast lysates with anti R453W (panel I), anti R482W (panel II), anti Lamin A/C 4C11 (panel III), and anti β-actin (panel IV). (B) Immunofluorescence of wild-type and EDMD primary human fibroblasts. Cells were stained with anti R453W (top panels) and anti Lamin A/C 4C11 (bottom panels). The DNA was counterstained with Hoechst 33342. Bar scale, 20 µm. (C) Immunofluorescence of wild-type and FPLD primary human fibroblasts. Cells were stained with anti R482W (top panels) and anti Lamin A/C 4C11 (bottom panels). The DNA was counterstained with Hoechst 33342. Bar scale, 20 µm. (D) Immunoprecipitation of wild-type and mutant lamin A/C from wild-type, EDMD, and FPLD cell lysates. The precipitated proteins were separated by 7.5% SDS-PAGE and analyzed by Western blotting with either the anti R453W or anti R482W antibody (top panels), or with anti Lamin A/C 4C11 (bottom panels).