Fig. 2. Nesprin-3 interacts with Sun proteins through its PPPX consensus sequence. (A) PA-JEB cells stably expressing GFP-nesprin-3 or GFP-nesprin-3^PPPT were fixed in paraformaldehyde, stained for endogenous Sun1 and Sun2 and analyzed by confocal microscopy. Bar, 10 µm. (B) PA-JEB cells stably expressing GFP-nesprin-3^PPPT were fixed in paraformaldehyde, stained for endogenous plectin (HD-121 antibody), the ER (PDI antibody) or the Golgi complex (Golgin97 antibody) and analyzed by confocal microscopy. Bar, 10 µm. (C) Ultrathin sections of PA-JEB cells stably expressing GFP-nesprin-3 or GFP-nesprin-3^PPPT were labeled with a pAb against GFP, followed by an incubation with 10 nm colloidal-gold-conjugated protein A. Nesprin-3 associates with the ONM (arrowhead) and the ER (arrow). N, nucleus; C, cytoplasm; NE, nuclear envelope; ER, endoplasmic reticulum. Bars, 200 nm. (D) Whole-cell lysates (WCLs) from PA-JEB cells stably expressing GFP-nesprin-3 or GFP-nesprin-3^PPPT were immunoprecipitated with the nesprin-3 mAb Nsp3. Immunoprecipitates (IPs) and WCLs were probed with antibodies directed against Sun1, Sun2, nesprin-3, and lamins A and C.