Mms 120r

The following primary antibodies were used for immunofluorescence microscopy: human anti-centromere (CREST serum 1:3, Antibodies Inc., 15-234-001; Antibodies Inc., Davis, CA, USA), mouse monoclonal anti-lamin A/C (1:30; Abcam, ab40567; Abcam, Cambridge, UK), monoclonal anti-LAP2α (1:10; clone 15/2, kind gift of Dr. Roland Foisner, Max F. Perutz Laboratories, Vienna, Austria), monoclonal antibody mAb414 (1:2000; Covance, MMS-120R; Covance, Emeryville, CA, USA), monoclonal anti-Hec1 (1:200; clone 3G9, Abcam, ab3613), and rabbit polyclonal anti-LAP2α (1:1000; Abcam, ab5162), polyclonal anti-lamin A (1:500; Sigma-Aldrich, L1293; Sigma-Aldrich, Diegem, Belgium), polyclonal lamin B1 (Abcam, ab16048), polyclonal anti-Sun1 (1:1000, kind gift of Dr. Ulrike Kutay, ETH Zurich, Switzerland), polyclonal anti-Sun2 (1:100; Sigma-Aldrich, HPA001209), polyclonal anti-emerin (1:1000; Bethyl Laboratories, A304-491A; ImTec Diagnostics, Antwerpen, Belgium), as well as polyclonal anti-Nesprin-2 (1:50, kind gift of Dr. Iakowos Karakesisoglou, Durham University, UK). Secondary antibodies were the corresponding goat anti-mouse IgG Alexa 568 (1:1000; Invitrogen), goat anti-mouse IgG Alexa 555 (1:1000; Invitrogen), goat anti-rabbit IgG Alexa 568 (1:1000; Invitrogen), goat anti-mouse IgG Alexa 633 (1:350; Invitrogen), and goat anti-rabbit IgG Alexa 633 (1:350; Invitrogen).

Antibodies used in this study include: mouse anti-RanGAP1 (19C7) monoclonal antibody (mAb) [14 (link)] from Dr. Michael Matunis (Johns Hopkins University, Baltimore, MD); mouse anti-Myc (9E10) mAb (sc-40; Santa Cruz); rabbit anti-Myc polyclonal antibody (2272; Cell Signaling); rabbit anti-RanBP2 polyclonal antibody (ab64276; Abcam); mouse mAb414 (MMS-120R; Covance); mouse anti-CRM1 mAb (611832; BD Biosciences).

Xenopus S3 cells were grown in 70% Leibovitz L-15 media GlutaMAX Supplement, 10% FBS and 500 units/ml penicillin-streptomycin (all from Gibco). Cells quickly washed with 70% PBS supplemented with 0.1 (v/v)% Triton X-100 and fixed for 5 min in 2% paraformaldehyde supplemented with 0.5 (v/v)% Triton X-100. Immunofluorescence analysis was performed as described17 (link). The rabbit polyclonal antibody was used in a 1:100 dilution and visualized by an Alexa 488-conjugated goat anti-rabbit antibody (Invitrogen) diluted 1:2,000. Mab414 ascites (Covance, MMS-120R), which marks nuclear pore complexes, was used in a 1:2,000 dilution and visualized by a Cy3-conjugated goat anti-mouse antibody (Invitrogen) diluted 1:2,000. Chromatin was stained with 10 μg/ml DAPI. Fluorescence images were acquired using a confocal microscope (LSM780, Zeiss) using 405-, 488-, and 561-nm laser lines and an apochromat 63 × NA 1.40 oil DIC M27 objective.