Fig. 5. Plectin dimers are required for the interaction with nesprin-3 at the NE. (A) Schematic domain structure of a plectin molecule, containing an ABD (purple), a plakin domain (green), a rod domain (yellow) and a series of plakin repeats (blue). The length of the different plectin constructs used and their respective domains are indicated below. (B) PA-JEB cells stably expressing GFP-nesprin-3 or GFP-nesprin-3 were transiently transfected with HA-tagged constructs encoding plectin 1-399, plectin 1-606, plectin 1-2532 or full-length plectin. 48 hours after transfection, cells were fixed in paraformaldehyde, stained for HA and analyzed by confocal microscopy. Bar, 10 µm. (C) PA-JEB, MD-EBS-1 and MD-EBS-2 keratinocytes were lysed and WCLs were analyzed by western blot for the expression of plectin (clone 31). Myosin heavy chain (MHC) served as a loading control. (D) PA-JEB cells expressing GFP-nesprin-3 and MD-EBS-1 cells stably expressing GFP-nesprin-3 or GFP-nesprin-3 were fixed in paraformaldehyde, stained for plectin (clone 31) and analyzed by confocal microscopy. Arrows indicate the presence of plectin at the NE. Bar, 10 µm.