Sun3AB-3’Xho), digested with the respective enzymes and cloned into NdeI/XhoI sites of pET-21a(+) (Novagen). Similarly, a Sun1 fragment coding for amino acids 427-722 was amplified from full- length Sun1 (Sun1AB-5’Nde; Sun1AB-3’Eco) and cloned into NdeI/EcoRI sites of pET-21a(+) [68]. To generate Myc-tagged constructs for immunoprecipitation, PCR fragments of Sun3 coding for the full-length protein (Sun3FL: Sun3-5’Eco; Sun3- 3’Sal) and Sun1 coding for amino acids 501–913 (Sun1TM+Lum: Sun1IP-5’Eco; Sun1IP-3’Sal) were cloned into pCMV-Myc vector (Clontech Laboratories). To create GFP-tagged KASH domains either of Nesprin1 or Nesprin3, Nesprin1TM+KASH (74 C- terminal amino acids of murine Nesprin1) and Nesprin3TM+ KASH (67 C-terminal amino acids of Nesprin3) fragments were amplified by RT-PCR from NIH/3T3 fibroblasts. PCR products were cloned into the SmaI site of pEGFP-C2 (Clontech). Expression analysis by RT-PCR Expression profiles of Sun1 and Sun3 were analyzed by RT- PCR using total RNA, isolated from testes of adult mice as well as from testicular suspensions of pubertal mice (8–25 days). Reverse transcription was performed on 1 mg of total RNA as described above. To monitor Sun1 RNA expression that includes predicted N-terminal variable isoforms [4,12] we used a 5’-oligonucleotide corresponding to a sequence in exon 6 (Sun1Exp-5’) and a 3’- oligonucleotide, complementary to the sequence in exon 11 (Sun1Exp-3’). To verify testis-specificity and expression profile of Sun1g a second primer pair that specifically amplifies Sun1g was applied (Sun1ATG-5’; Sun1g-3’). Sun3 expression was analyzed by using primers Sun3AB-5’Nde and Sun3AB-3’Xho. Antibodies For the generation of specific antibodies against Sun1 and Sun3, His-tagged Sun1 and Sun3 fusion constructs were expressed in E. coli RosettaTM (Novagen), purified under denatured conditions on Ni-NTA agarose resin (Qiagen) and used for immunizing of guinea pigs (Seqlab, Go¨ ttingen, Germany). Obtained sera were affinity purified on HiTrapTM NHS-activated HP Columns coupled with the respective antigens (GE Healthcare) as described by the manufacturer, and tested for specificity by immunofluor- escence microscopy and immunoblotting. Further antibodies used in this study were: affinity purified rabbit anti-lamin B3 [42]; affinity purified rabbit anti-Cage1 [48]; monoclonal antibody 13d4 against LAP2 [43]; affinity purified rabbit anti-SYCP3 [50]; and a rabbit polyclonal antibody against Nesprin3 (kindly provided by Dr. Arnoud Sonnenberg, The Netherlands Cancer Inst., Am- sterdam, The Netherlands) [18]. Rabbit anti-SYNE1 (HPA019113) was purchased from Atlas Antibodies. Monoclonal antibodies against a-Tubulin (T5168) and b-Tubulin (T4026) were obtained from Sigma-Aldrich. Monoclonal mouse anti-GFP (B-2) was purchased from Santa Cruz Biotechnology. Mouse mono- clonal anti-myc antibody (R950-25) was obtained from Invitrogen. Secondary antibodies coupled to Cy2 and Texas Red as well as peroxidase-conjugated secondary antibodies were obtained from Dianova. Immunocytochemistry For immunostaining, testes of adult mice were fixed for 3 hours at room temperature in PBS containing 1% paraformaldehyde, embedded in paraffin and sectioned (sections 3–10 mm). Paraffin removal and antigen retrieval was performed as previously described [48]. After permeabilization with PBS containing 0.1% Triton X-100 for 10 minutes sections were blocked for 1 hour with PBT (0.15% BSA, 0.1% Tween 20 in PBS). Then sections were subjected for 1 hour to respective primary LINC in Sperm Head Formation antibodies. After washing twice in PBS, specimens were incubated with appropriate secondary antibodies conjugated to Cy2 or Texas Red. DNA was stained with Hoechst 33258 (Hoechst). Digital images were taken by confocal microscopy with a Leica TCS-SP2 AOBS confocal laser scanning microscope (63x/1.40 HCX PL APO oil-immersion objective, pinhole at 1 P AU; Leica). All images shown in this study are maximum 2D projections calculated either of three or of all sequenced single sections. Image data were processed using Adobe Photoshop and AmiraH Software. Cell culture and transfection HeLa cells were grown in 10% bovine growth serum, 1% L- glutamine, 1% penicillin/streptomycin with DMEM (Invitrogen). Cells were cotransfected with the respective pEGFP- and pCMV- Myc- expression vectors using EffecteneTM transfection reagent (Qiagen) according to manufacturer’s instructions and incubated for 24 hours at 37uC and 5% CO2 content. Co-immunoprecipitation Co-immunoprecipitation was performed as described by Stewart-Hutchinson et al. [5]. HeLa cells grown to confluency on 35-mm dishes were harvested 24 hours post transfection with the respective constructs and lysed with 500 ml of radio- immunoprecipitation assay (RIPA) lysis buffer (1% Triton X- 100, 0.5% sodiumdesoxycholate, 0.1% SDS, 1 mM b-glyceropho- sphate, 1 mM Na3VO4, 1 mM EDTA, 1 mM EGTA, in PBS, protease inhibitor mixture). Lysates were cleared by centrifugation at 15.000g in a microfuge at 4uC for 15 minutes and the supernatant was divided into 250 ml aliquots. Each aliquot was then incubated with either 1.5 mg monoclonal mouse anti-myc antibody (Invitrogen) or mouse IgG unspecific antibody (Dianova). After overnight incubation at 4uC with continuous shaking, immune complexes were pulled down with DynabeadsH Protein G (Invitrogen): 20 ml of the magnetic beads were added, incubated for 2 hours at 4uC, harvested by magnet and washed three times with 250 ml RIPA buffer. Finally the beads were resuspended in 2xSDS-sample buffer (120 mM Tris/HCl, 10% SDS, 20% Glycerol, 20% 2-mercaptoethanol, bromphenol blue, pH 6.8), heated to 95uC for 15 minutes and resolved by SDS-PAGE. SDS-PAGE and Western blotting Protein samples resulting from immunoprecipitation experiment, samples of different mouse tissues as well as testicular cells from mice of different ages (8–25 days) were resuspended in 2xSDS-sample buffer and heated to 95uC for 15 minutes. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked over night at 4uC in TBST buffer (10 mM Tris/HCl, 150 mM NaCl, 0.1% Tween 20, pH 7.4) containing 10% milk powder and incubated for 1 hour at room temperature with respective primary antibody in blocking solution. After washing with TBST buffer, membranes were incubated with appropriate secondary antibodies coupled to peroxidase. Signals were detected using the Western LightningH Plus-ECL, Enhanced Chemiluminescence Substrate (Perkin Elmer). Supporting Information Table S1 Oligonucleotides used in the study. Found at: doi:10.1371/journal.pone.0012072.s001 (0.06 DOC) MB Figure S1 Sun3 expression during mammalian spermiogenesis. Localization of Sun3 within seminiferous tubules was analyzed by PLoS ONE | www.plosone.org 11 August 2010 | Volume 5 | Issue 8 | e12072