Fig. 7. (A) Schematic representation of the positions of the five peptides used for generating polyclonal antibodies against nesprin-1. The peptides N1 and N2 were specific to nesprin-1ß but did not produce functional antibodies. The small black boxes indicate peptides. (B) In vitro transcription and translation of human nesprin-1
cDNA. The expression vector pcDNA3.1 containing the full-length cDNA of human nesprin-1 (112 kDa), the luciferase (61 kDa) T7 control cDNA as a positive control, the TNT lysate reaction without DNA as negative control. (C) Immunoprecipitation of human nesprin-1 generated from radioactive in vitro transcription/translation using antibodies C1 that recognised a 112kDa protein and N1, a non-specific negative control. (D) Western blot of human nesprin-1-EGFP fusion protein in transfected COS-7 cells. Control; untransfected COS-7 cells, GFP: pEGFP-C1 vector alone and nesprin 1-pEGFP-C1. (E) Western blot of human VSMC (I) and peripheral blood leukocyte (II) cell lysates using antibodies N3 and C1. The 112 kDa and 380 kDa bands are indicated by arrows. (F) Immunoprecipitation of C2C12 cells (I) using antibodies N3 and C1. Rabbit IgG as negative control. Western blot of C2C12 whole cell lysates (II) indicating that many of the IP products are also identified on western blots. (G) Immunofluorescent staining of nesprin-1 in mouse C2C12 myoblasts and human VSMCs. Endogenous nesprin-1 (green) was detected by nesprin-1 antibodies (N3, C1 and C2) and visualised by confocal microscope. DAPI is shown as false colour red in composite.
