Nesprin-1 and Nesprin-2 Immunofluorescence and Western Blotting

Nesprin-1 polyclonal antibodies (SpecII) and mAb K58–398–2 directed against the C-terminus of human Nesprin-1, affinity-purified rabbit anti-Nesprin-1 ABD, and mAb K43–322–2 directed against the N-terminus were used.²² (link) pAbK1 was used to detected Nesprin-2.⁶ (link) Immunofluorescence and western blotting were done as described.²³ (link) Antibodies used were specific for Emerin (4G5, Abcam), LAP-2 (BD Transduction Laboratories), Lamin B1 (Abcam), Lamin A/C (StCruz), SUN1 (Abcam), SUN2 (Abcam), mAb414 recognizing nuclear pore complex (NPC) proteins (Abcam), anti-phospho-Ser139 H2AX (Millipore), anti-phospho-Ser317 Chk1 (Cell Signaling), anti-phospho-Ser345 Chk1 (Cell Signaling), anti-phospho-Thr68 Chk2 (Cell Signaling), Ku70, MSH2, MSH6 (all Abcam), and appropriate secondary antibodies conjugated to Alexa 488/568 (Invitrogen). Imaging was done by confocal laser scanning microscopy (Leica TCS-SP5). Images were processed using TCS-SP5 software.