Fig. 5a–c.
SUN-protein dominant-negative (DN) interference affects KLC1 NE localization. NIH-3T3 fibroblasts transiently expressing SPGFP control (a) and DN-SUNL constructs (b) were subjected to indirect immunofluorescence using anti-nesprin-2 (Nes2NT, mAb K56-386) and anti-KLC1 (pAb H-75). The nesprin-2 and KLC1 NE pattern is unaffected in control fibroblasts (a, arrows). In DN-SUNL-expressing cells (b, asterisk), however, staining of both nesprin-2 and KLC1 NE is diminished (b, arrows). b′ The histogram represents a statistical evaluation of the KLC1 localization phenotypes at the NE in control and DN-SUNL-expressing cells (results are the mean ± SD; Student’s t test: P normal = 3.84 × 10−4, P reduced = 6.46 × 10−3, P absent = 1.79 × 10−2). b″ Immunoblot analysis of stably transfected DN-SUNL human keratinocytes (HaCaT; clones #1 and #2) and untransfected HaCaT control cells. Note that the DN-SUNL expression did not alter the nesprin-2, KLC1, and tubulin expression levels. Actin is shown as a loading control. c Untransfected NIH-3T3 fibroblasts exhibiting weak nesprin-2 staining (asterisk) have a decreased KLC1 immunostaining at the NE, whereas an intensive nesprin-2 signal correlates with strong KLC1 staining at the NE (arrows). DAPI was used to visualize DNA. c′ KLC1 and tubulin stainings of colchicine-treated NIH-3T3 cells indicate a direct correlation of their fluorescence intensities at the NE (arrows denote weak staining signals, whereas strong signals are labelled by arrowheads). Scale bars 10 μm