Nuclear positioning was affected in TA muscles from nesprin 1α2 KO mice. (A) TA skeletal muscle fibers isolated from either WT or nesprin 1α2 KO (N1α2 KO) mice were fixed and stained using Lamin A/C (A/C), Lamin B1 (B1), Sun1, Sun2, emerin (green), andαα-actinin (red) antibodies. Note the striking differences in nuclear positioning between WT and KO fibers but similar localization of the LINC-associated components (arrowheads). Sun1 localization was more cytoplasmic in KO TAs compared with controls (yellow arrows). (B) Quantification of internuclear distances of nesprin 1α2 KO (green bars) and WT mice (blue bars) shown in A. Note the striking clustering of nuclei in nesprin 1α2 KO myofibrils. n = 220–436 and n = 201–226 internuclear distances were counted for nesprin 1α2 KO and WT, respectively. (C) Western blots of the LINC complex proteins Sun1 and Sun2, the LINC-associated protein emerin, and the nuclear lamins A/C, B1, and B2 from WT and nesprin 1α2 KO TA muscle lysates. Note that the levels of most of the proteins were unaffected except Sun1, which was down-regulated. GAPDH served as a loading control. (D) Low-magnification representative images of those shown in A. n = at least three to four pups per genotype for each panel. Bars: (A) 5 µm; (D) 10 µm. DIC, differential interference contrast.