were pre-stimulated with two portions of 150 iU of HCG (Human Chorionic Gonadotropin; Sigma) 48 and 24 hours before sperm isolation. Xenopus sperm chromatin binding assay Isolated and demembranated sperm chromatin (DSP) from Xenopus was used in the condensed form and in the decondensed form prepared by incubation of sperm chromatin with poly L- glutamic acid in Pfaller buffer [70] for 20 min at room temperature. Sperm chromatin decondensation was performed as follows: chromatin of about 105 sperm was incubated in Pfaller buffer with 0.5 mg/ml poly L-glutamic acid for 20 min at room temperature in a total volume of 40 ml. For sperm chromatin binding assays the bacterially expressed proteins (0.25 mg–1 mg) were added to the condensed or decondensed chromatin (about 105) in extraction buffer in a total volume of 50 ml. The reaction proceeded for 15 min at room temperature. Chromatins were recovered on glass coverslips and processed for immunofluorescence analyses according to Drummond et al. [71]. Images were taken using an inverted Olympus IX70 fluorescence microscope. Work with animals The Laboratory of Nuclear Proteins has all necessary documents, licenses and the Local Ethical Committee license for work with Xenopus laevis. The Local Ethical Committee License for experiments with Xenopus is permanent and was issued on 02.07.2003 (Number of application: 216/03; Number of license: 75/03). The Polish National Ethical Committee (KKE) issued a license to the Faculty of Biotechnology, University of Wrocław on behalf of the Laboratory of Nuclear Proteins for animal maintenance and performing experiments on animals (License Number KKE/36/2007/pl; Issued 14.03.2007). The Laboratory of Nuclear Proteins has certification from the Polish Ministry of Science and Higher Education for cultivation of animals and performing research (Decision No. 9/2007; Identification number 110). Xenopus in vitro sperm pronuclei assembly Xenopus sperm pronuclei were reconstituted according to Salpingidou et al. [69]. In tests with bacterially expressed lamins added to the reaction, typically between 0.25 mg and 1 mg of protein was added. Assembly reactions were visualized with an Olympus IX70 fluorescence microscope or Olympus FV500 laser confocal microscope. Lamin Dm staining in tissue cultured cell HeLa cells or Xenopus XTC cells were grown on 12 mm cover slips (in a 50 mm culture dish plate) up to 80% confluency as described previously [72]. S2 cells were cultured as described previously [37–38]. Cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 in PBS, and after washing with PBS were incubated for 20 minutes at room temperature with 25 ml of PBS containing 0.5 mg of lamin. After incubation, cells were washed, fixed and processed for immunofluorescence. Primary antibodies were used for human lamin C (affinity purified anti-lamin C specific fragment), Drosophila lamin Dm (ADL 67) and lamin C (ALC 28). The HeLa cell line was obtained from Ludwik Hirszfeld Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences in Wrocław. The XTC cell line was obtained from the Integrative Cell Biology Laboratory, School of Biological and Biomedical Sciences, University of Durham, UK. The Function of Drosophila Lamins Phosphorylation Nuclei isolation Human cells or Xenopus XTC cells were trypsinized, centrifuged at 1186 g for 5 min in 50 ml Falcon tubes and resuspended in NIB buffer nucleus isolation buffer (10 mM Tris HCl pH 7.6; 10 mM NaCl; 3 mM MgCl2; 0.05% Triton X-10; with protease inhibitors) 1 ml/106 cells. After 15 minutes on ice, cells were homogenized and centrifuged through 30% sucrose in NIB in Eppendorf tubes (20006 g, 10 min, 4uC). Cell nuclei were suspended in NIB buffer. Isolation of cell nuclei from Drosophila embryos and S2 cells was described previously [37,39]. Lamin binding to isolated cell nuclei or chromatin Between 1 and 0.25 mg of each protein was added to the 105 of cell nuclei or equal amounts of chromatin in 20 ml of PBS and incubated for 20 min at room temperature. Fixation was in 100 ml of 1% EGS in PBS for 30 min at 37uC. Samples were layered over 30% glycerol in PBS-Tween (0.05%) and centrifuged onto 22 mm coverslips (20006g, 10 min, 4uC), fixed with formaldehyde and processed for immunofluorescence. Antibodies Donkey anti-rabbit-FITC conjugated and donkey anti-mouse- TRITC conjugated (Jackson ImmunoResearch) were used at a dilution of 1:50. ADL67 mouse monoclonal anti-lamin Dm was used at a dilution of 1:2. LC 28 mouse monoclonal antibodies against lamin C were used at a dilution of 1:3. R389 rabbit serum against lamin C was used at a dilution of 1:50 for IF. Affinity purified antibodies for bacterially expressed human lamin C were used at 1:20 dilution. Affinity purified IgGs from rabbit serum against lamin Dm were used at a dilution of 1:50 [73]. CD spectra measurement CD (circular dichroism) spectra were collected with a J-175 spectropolarimeter (Jasco) using 2 nm bandwidth and response time of 1 s. Sampling was every 1 nm. Each curve represents the medium value for 3 scans. All experiments were carried out using cuvettes of 0.1 cm path length. Ellipticity was expressed as mean residue ellipticity [h] in deg cm2 dmol21 using the following equation: [h] = [hobs/(106lxc)]/n, where hobs is the observed ellipticity in degrees, l is the optical path length of the cell in cm, c is the protein molar concentration, and n is the number of residues in the protein. Measurements were performed in phosphate buffer (20 mM sodium phosphate buffer with 0.6 M urea, pH 8.0). Transfection and analyses HeLa cells (human epithelial carcinoma cell line) were transfected by plasmids encoding lamins by using Metafectene reagent (Biontex Laboratories GmbH, Germany) according to the manufacturer’s instructions. S2 cells (Drosophila Schneider 2 cells, Invitrogen) were transfected by electroporation with CLB- Transfection Device (Lonza), program CELL5, using approx. 107 cells and 10 mg of plasmid DNA. Transfection efficiency was about 30–50% in the case of HeLa cells and up to 10% in S2 cells. Based on calculations performed on transfected HeLa, S2 and HEK293 cells (90% efficiency of transfection) followed by western blot and IF analyses, it is possible to estimate that most of the transfected HeLa cells expressed exogenous lamins at a compa- rable amount to endogenous lamins. It was similar in S2 cells. For analysis of data on lamin location we counted cells with location of transfected fusion lamin proteins in nucleus only, in nucleus and cytoplasm, and in cytoplasm. For nuclear location we assumed that at least 75% of the label was in the cell nucleus. For mixed, nuclear and cytoplasmic location we assumed that the label PLoS ONE | www.plosone.org 12 February 2012 | Volume 7 | Issue 2 | e32649