in nucleus and cytoplasm was lower than 75%. For cytoplasmic location we assumed that at least 75% of the fusion protein label was in the cytoplasm. The data for location of lamin fusion proteins in nuclear membrane, inclusions, diffused through nucleus or cytoplasm, or with mixed phenotype were based on the following assumptions: For counting cells exclusively in the nuclear membrane and inclusion categories, at least 65% of the label had to be in these locations respectively. As diffused phenotype cells were counted cells with predominantly diffused or dispersed label but also ‘‘mixed’’ phenotype – cells with the label in the nuclear envelope and/or inclusions but without obvious domination of either phenotype. The rationale for assigning such phenotypes as diffused was that a significant fraction of the label in such cells is frequently also ‘‘dispersed’’. FRAP analysis FRAP analyses were performed on transfected HeLa, XTC and S2 tissue cultured cells. HeLa cells (in MEM alpha supplemented with Glutamax) were previously adapted to grow at room temperature without carbon dioxide. XTC cells and S2 cells normally grow at room temperature without carbon dioxide. Cells were cultured on glass coverslips for 24 h and 48 h after transfection and analyzed under a 406 (water immersion) objective in culture medium under a Zeiss LSM 510 Meta microscope according to the manufacturer’s instructions. For each mutant FRAP analyses were performed on lamin proteins located in the nuclear interior, nuclear envelope and cytoplasm (if available). Mobility analyses were performed many times for each compartment. For calculations, at least three measurements, of the best quality from each compartment were selected. We used FRAP analysis software (Zeiss LSM510 software - ZEN 2008) for experiment setup and data collection (prebleach imaging, laser pulse, recovery imaging, collection of data, etc.) and calculation of values for the mobile fraction (Mf measured in %) and time required for 50% fluorescence plateau recovery of the bleach zone (t1/2 measured in seconds), using normalized fluorescence intensity. The diffusion coefficient [D measured in mm2/s] was determined from the equation D = v2/4t1/2, where v is the radius of circular ROI and t1/2 is diffusion half time. FRAP experiments were performed on cells transfected with wild type human lamin A, B1 and C, wild type lamin Dm and pseudopho- sphorylation mutants fused to EGFP protein. Data demonstrated in Figure 7 were collected with lamin Dm farnesylation incompetent mutants since lamin Dm and all pseudophosphoryla- tion mutants with wild type farnesylation sequence showed the same mobility and the same percentage of mobile fraction. Supporting Information Figure S1 Overexpression and purification of lamin Dm, lamin C and mutant proteins. 12% SDS-polyacryl- amide gel electrophoresis of fractions from the purification of wild type lamin Dm and Lamin Dm S45E (Panel A and B respectively) together with lamin C and mutant lamin C S37E (Panel C and D respectively) and all purified lamins (Panel E). The lamin proteins were overexpressed in E. coli BL21DE3(pLysS) bacterial cells. Bacteria were lysed and proteins were solubilized from inclusion bodies in buffer containing 6 M urea and purified by metal affinity chromatography on Ni-NTA-agarose columns. Please note that relative electrophoretic mobility of particular protein may vary The Function of Drosophila Lamins Phosphorylation from gel to gel and lane to lane due to the different proteins concentration, buffer composition, position etc. Panel E illustrates the quality of purified lamin proteins. M – Prestained Protein Molecular Weight Marker (Fermentas), BSA (bovine serum albumin) – 2.5 mg, S4 – proteins extracted in buffer containing 6 M urea, U – unbound fractions, E – elution fractions. (TIF) Figure S2 Circular dichroism (CD) spectra of the purified wild type lamin C and lamin Dm, and their mutants. Curves representing different temperatures are marked by different colours. Initial curves on both panels have two peaks, one at 208 nm and the second at 220 nm, demonstrating typical shape for native proteins containing a large proportion of a-helical structure. The denaturation was found to be largely reversible (R - renatured CD spectra). (TIF) Figure S3 All lamin Dm proteins bind to chromatin and nuclear envelope in in vitro nuclear assembly system from Xenopus. In vitro Xenopus sperm pronuclei assembly reaction was used to assess the ability of lamin Dm mutants to bind to assembling chromatin and nuclear envelope structures. Assembly reaction was carried out for 40 min in the presence of bacterially expressed proteins. (TIF) Figure S4 Mutant lamin C S37E protein does not bind to in vitro assembled Xenopus sperm pronuclei. In vitro Xenopus pronuclei assembly reaction was used to assess the ability of bacterially expressed lamin C and lamin C S37E mutant to bind to assembling chromatin and nuclear envelope structures. The control experiment was without addition of any exogenous protein. Assembly reaction was carried out for 30 min in the presence or absence of bacterially expressed proteins. (TIF) Figure S5 Localization of fusion EGFP: lamin A, lamin B and lamin C 48 hours post-transfection into HeLa cells visualized under confocal microscope in order to visualise differences in phenotype depending on fusion protein expression level. Cells were stained for DNA with DAPI and for lamin C with rabbit affinity purified antibodies. Staining with secondary antibodies was with goat anti-mouse secondary antibodies conjugated with TRITC and goat anti-rabbit secondary antibodies conjugated with Cy-5 respectively. Particular lamin fusion proteins were visualized by EGFP fluorescence. Single confocal Z-sections are shown through the center of nuclei. Only the most typical phenotypes are shown. (TIF) Table S1 The content of distinct secondary structure types present in analyzed lamins and residue molar ellipticity. The content of distinct secondary structures in lamin proteins was calculated using CDFIT software. Molar ellipticity values were measured at 20uC in PBS with 0.6 M urea. (DOC) Author Contributions Conceived and designed the experiments: MZC RR. Performed the experiments: MZC KP KW KK MDM. Analyzed the data: MZC RR. Wrote the paper: RR. PLoS ONE | www.plosone.org 13 February 2012 | Volume 7 | Issue 2 | e32649