Fig. 6. Expression of the plectin ABD stabilizes the actin cytoskeleton and has a dominant-negative effect on the localization of endogenous plectin at the NE. (A) PA-JEB cells stably expressing GFP-nesprin-3 were transiently transfected with constructs encoding HA-plectin ABD or HA-plectin ABD^E95S. 48 hours after transfection, cells were fixed in paraformaldehyde, stained for HA and endogenous plectin (HD-121 antibody) and analyzed by confocal microscopy. Bar, 10 µm. (B) PA-JEB cells stably expressing GFP-nesprin-3 were transiently transfected with a construct encoding the HA-plectin ABD. The cells were treated with cytochalasin D for 30 minutes, fixed in paraformaldehyde and stained for HA and either endogenous plectin (HD-121 antibody) or F-actin. Bar, 10 µm. (C) PA-JEB cells stably expressing GFP-nesprin-3 were treated with 100 nM jasplakinolide for two hours. Treated and untreated cells were fixed in paraformaldehyde and stained for F-actin and endogenous plectin (HD-121 antibody). Bar, 10 µm.