were fixed in 4% paraformaldehyde (PFA) in PBS at 4uC overnight. Animal studies were carried out under regulations of the Animals (Scientific Procedures) Act 1986 of the UK Government, and in according with guidance issued by the Medical Research Council, UK in Responsibility in the Use of Animals for Medical Research (July 1993). Two-dimensional gel electrophoresis (2-DE) Samples, comprising whole embryos (n = 10 of each genotype) or individual caudal regions (n = 10 of each genotype), were prepared by sonication in lysis buffer as described previously [55]. Proteins were separated by isoelectric focussing on pH gradients of pH 4–7 or 3–5.6, followed by SDS-PAGE on 12% polyacrylamide gels, as described [56]. Gels were fixed and stained using PlusOne silver stain (GE Healthcare) and scanned using a GS-800 calibrated densitometer (BioRad). Gel images were analysed using Progenesis SameSpots (Non-linear Dynamics) with separate between-genotype comparisons for whole embryos (n = 5 pH 4– 7 and 5 pH 3–5.6 gels for each genotype) and caudal regions (n = 5 pH 4–7 and 5 pH 3–5.6 gels for each genotype). Liquid chromatography electrospray tandem mass spectrometry (LC-ESI-MS/MS) Protein spots were excised manually from a minimum of four different gels, so that each spot was analyzed at least in quadruplicate, subjected to in-gel digestion with trypsin and analyzed by LC- ESI-MS/MS (QToF-micro; Waters Corp.) as described previously [55]. Mass spectrometry data were searched against the SwissProt database using the MASCOT search algorithm (Matrix Science, London, UK). One missed cleavage per peptide was allowed. Sequence analysis Genomic DNA fragments spanning exons of Lmnb1 were amplified by PCR (see Table S4 for primer sequences). Purified PCR products were sequenced using big dye terminator chemistry (Applied Biosystems) and analysed on a MegaBACE 1000 (Amersham). Sequence reads derived from both strands were assembled, aligned and analysed for nucleotide differences using Sequencher (GeneCodes). Western blot Protein lysates (1 mg per lane) in RIPA buffer were run on 10% Bis-Tris gels (NuPage, Invitrogen) and transferred to PVDF membrane (XCell II Blot Module, Invitrogen). Immunodetection was performed by standard methodology using antibodies to lamin B1 (S-20) and b-tubulin for normalisation (primary antibodies from Santa Cruz Biotechnology and used at 1:1000). Proteins were detected using horseradish peroxidise-conjugated secondary anti- bodies (DAKO), followed by development with ECL plus Western blotting detection system (GE Healthcare). Films were scanned on a GS-800 Densitometer (Bio-Rad) for quantification. Quantitative real–time RT–PCR RNA was purified (TRIzol Reagent, Invitrogen) from isolated caudal regions of E10.5 embryos or from MEFs, genomic DNA removed by DNase I digestion (DNA-free, Ambion) and first strand cDNA synthesis carried out (SuperScript II, Invitrogen). qRT-PCR was performed (MESA Blue Mastermix for SYBR Assay, Eurogentec) on a 7500 Fast Real Time PCR system (Applied Biosystems), with each sample analysed in triplicate. Primers for Lmnb1 were designed to amplify a 221 bp product (nucleotides 1267–1487 of coding sequence (Ensembl NM 010721.1; ENSMUSG00000024590). Additional primer pairs were: cyclin A2 (Ccna2) 59-CATGTCACTGCTGGTCCTTC Lamin B1 and Neural Tube Defects and 59- TGATTCAAAACTGCCATCCA); cyclin B1 (Ccnb1) 59- GGAAATTCTTGACAACGGTG and 59-TGCCTTTGTC ACGGCCTTAG; Cyclin D1 (Ccnd1) 59-GCGTACCCTGACAC- CAATCT and 59-CTCTTCGCACTTCTGCTCCT; Smc2 59- AAATAGCCGCCCAGAAAACT and 59-GAGCGTTCCTT GGTGTCTTC. Primers for p16Ink4a were described previously [27]. Results were normalized to Gapdh as described previously [9]. For microarray, RNA was further purified using the RNeasy Micro Kit (Qiagen), followed by cDNA synthesis, linear amplifica- tion and labelling of cRNA using GeneChip 39IVT Express Kit (Affymetrix). RNA and cRNA quantity and quality were determined by Nanodrop spectrophotometer and Bioanalyser 2100 (Agilent). Affymetrix Mouse 430_2 arrays were hybridised as standard (www. affymetrix.co.uk). Files were processed in GeneSpring GX (Agilent Technologies), with application of GC-RMA normalisation and Benjamimi-Hochberg multiple testing correction. Whole-mount in situ hybridisation Whole-mount in situ hybridisation, was performed as described previously [9], using a digoxygenin-labelled 561 bp cRNA probe which was complementary to nucleotides 726–1286 of the Lmnb1 transcript/coding sequence. Embryos were embedded in gelatine- albumin and sectioned at 50 mm thickness on a vibratome. Photobleaching experiments Constructs were generated in pcDNA3.1 vector by standard cloning methods, to express fusion proteins composed of a nuclear localisation signal, yellow fluorescent protein and full-length lamin B1 or C-terminal region. Plasmids were transfected into MEFs and FLIP was performed as described previously [51]. In brief, a region of interest (ROI) was photobleached at full laser power while scanning at 4% laser power elsewhere. For quantitative analysis, background intensity was subtracted, and intensities of a specific ROI outside the photobleached area were measured over time and normalized using intensities of an ROI in a transfected but non-bleached cell. Immunofluorescent labelling and laser-scanning confocal microscopy MEFs, derived from pools of 3–6 embryos at E13.5, were fixed in 4% PFA in PBS for 10 min, permeabilized with 0.4% Triton X- 100 in PBS for 5 min, and blocked with 0.4% fish skin gelatine in PBS for 30 min at room temperature. Incubations with primary and secondary antibodies were for 1 h each at room temperature. Primary antibodies were mouse anti-lamin B1 (8D1; [57] and rabbit anti-lamin A (ab26300: Abcam). Secondary antibodies were donkey anti–mouse and anti–rabbit (Jackson ImmunoResearch Laboratories) conjugated to Alexa Fluor 488 and Cy5 respectively. Imaging was performed using a confocal microscope (LSM 510 META; Carl Zeiss, Inc.) on an Axio Imager.Z1 (Carl Zeiss, Inc.) with a 636 1.4 oil immersion objective lens. Laser lines used were 405 nm, 488 nm and 633 nm to excite DAPI, Alexa Fluor 488 and Cy5, respectively. Fluorescence was detected using the following filters: base pairs 420–480, base pairs 505–530 and long pass 650. Images were analyzed using MetaMorph (MDS Analytical Technologies) or Image Browser (Carl Zeiss, Inc.) software. Cell cycle analysis MEFs were plated onto 13 mm cover slips (passage 3; 1.06105 cells per well in triplicate), cultured for 5 hours prior to addition of 10 mM EdU (Invitrogen). After 1 hour cells were fixed and processed for detection of EdU (Click-It EdU Imaging Kit). Cells were then washed in 0.1% Triton-X100 in PBS and blocked for PLOS Genetics | www.plosgenetics.org 11 November 2012 | Volume 8 | Issue 11 | e1003059