Cells were washed in PBS and mounted with VectaShield mounting medium with DAPI (Vector Laboratories). For double immunofluorescence for NUP62 and NUP133, cells were first incubated with NUP133 antibody, then washed and incubated with anti-mouse secondary antibody (Cy5), then washed and incubated with FITC-coupled NUP62 antibody (45 min). Epifluorescence microscopy was performed using a Zeiss Axioplan 2 microscope. Images were acquired with either 406(air) or 636(oil) objectives in z-stack series extending 10 to 15 um. Images were analyzed after 2D or 3D deconvolution (AutoQuant X; MediaCy- bernetics) and image stacks were projected on single planes. Confocal microscope was performed with the Leica TCS SP5 microscope, using the 488 nm and 543 nm lines of the conventional lasers. Stimulated emission depletion (STED) microscopy was performed with the Leica TCS STED microscope, using the 770 nm wavelength for depletion and the 635 nm pulse laser for excitation. All confocal and STED images were take using a 10061.4NA objective (oil). Images for confocal and STED microscopy were acquired in 8 to 10 um Z-stacks, and projected without deconvolution. Radial intensity distributions for nuclear sectors were defined and quantified using the Radial Profile Extended plug-in for Image J (Philippe Carl; University of Munster, Germany). Region of interest radii were set to extend from the local center of the nucleus to its boundary with the cytoplasm; radial extension integration angle was set at 20u (40u total), and intensity data was normalized to the length of each radial projection. Curve fitting by polynomial regression analysis and determination of confidence intervals was performed using TableCurve version 5.01 (Systat Software). All curves were fitted by polynomial regression to y = a+bx2+cx4+dx6+ex8+fx10+gx12+hx14+ix16+jx18+kx20. Immunoblotting Cell lysates were prepared by washing cells once in PBS followed by resuspension in SDS sample buffer (62.5 mM Tris- HCl, pH 6.8, 10% Glycerol, 2% SDS, 5% b-mercaptoethanol, 125 ug/ml bromophenol blue). Samples were heated to 90u for five minutes, cooled, and resolved by SDS-PAGE. Proteins were transferred to nitrocellulose sheets electrophoretically in transfer buffer (25 mM Tris, 92 mM glycine, 20% v/v methanol) and stored until use at 220u. Nuclear and cytosolic fractions were generated by the REAP method [43], which is rapid, minimizes exposure to hyperosmotic conditions, and preserves protein- protein intaractions. Briefly, cell monolayers are chilled on ice, scraped into PBS, and washed. One third of the samples is removed for the whole cell lysate. The samples are pelleted and lysed in an isotonic buffer containing 0.1% NP40, then pelleted. The supernatant is taken as the cytosolic fraction, the pellet is washed once in lysis buffer, and the washed pellet is dissolved in SDS sample buffer and used as the nuclear fraction. Samples were processed for SDS-PAGE as described above. Mmembranes were blocked for 2 hours at room temperature with 5% nonfat milk in TBS (25 mM Tris pH 7.4, 100 mM NaCl). The blots were then References 1. Schwartz TU (2005) Modularity within the architecture of the nuclear pore complex. Curr Opin Struct Biol 15: 221–226. 2. Alber F, Dokudovskaya S, Veenhoff LM, Zhang W, Kipper J, et al. (2007) The molecular architecture of the nuclear pore complex. Nature 450: 695– 701. 3. Lim RY, Fahrenkrog B (2006) The nuclear pore complex up close. Curr Opin Cell Biol 18: 342–347. 4. Lim RY, Ullman KS, Fahrenkrog B (2008) Biology and biophysics of the nuclear pore complex and its components. Int Rev Cell Mol Biol 267: 299–342. 5. Gillespie PJ, Khoudoli GA, Stewart G, Swedlow JR, Blow JJ (2007) ELYS/ MEL-28 chromatin association coordinates nuclear pore complex assembly and replication licensing. Curr Biol 17: 1657–1662. Diversity in Nuclear Pore Complexes incubated at 4u overnight in blotting solution (TBS supplemented with 5% nonfat milk, 1–5% horse serum, 0.5% Tween 20) with primary antibody (0.5–2.5 ug/ml). The blots were washed three times, and incubated with secondary antibody at room temper- ature in TBS/Tween 20 for 1.5 hours. Immunoreactive bands were visualized using enhanced chemiluminescence (ECL, Amer- sham) or with the Amersham ECL Plex immunoblotting system (GE Healthcare). The latter were scanned by the Typhoon Trio (GE Healthcare). Knockdown by siRNA transfection For siRNA knockdown experiments, cells were plated at 36104 per cm2 using 15% FBS in DMEM without addition of antibiotics for 24 hours. Cells were transfected 24 hours after plating with siRNA using Lipofectamine RNAiMAX (Invitrogen) and the manufacturers’ recommended procedures. The siRNAs for NUP62 knockdown (12–24 pmol) were purchased from Santa Cruz Biotechnology (product number sc-36107), and cells were analyzed 48–72 hours after transfection. The siRNAs for NUP214 knockdown (75 pmol) comprised a mixture of three custom siRNAs (59-GUGCAGGAAUUGAAGAAUAuu-39, 59-UCGA- CAAAGUGAUCAGAUUuu-39, and 59-GGGAGUUGUCGUA- GACUAUuu-39) combined with commercial siRNAs for NUP214 from Santa Cruz Biotechnology (product number sc-106320). Cells were incubated with the NUP214 siRNA mixture for 3 days and changed to new fresh media with new siRNAs for another 3 days before analysis. DNA-mediated transfections Plasmids containing the cDNA clones for human NUP62 and NUP214 were obtained from Open Biosystems. The V5 epitope tag (GKPIPNPLLGLDST) plus a strong Kozak consensus sequence and start codon were inserted in frame in place of the methionine start codons for NUP62 and NUP214 (insert: GCCACCATGGCGGGCAAACCGATTCCGAACCCGCTG- CTGGGCCTGGATAGCACC). The modified cDNAs were directionally cloned into the Not I and Sal 1 sites of pCMV- Sport-6 (Invitrogen). Cells were transfected using either Lipo293D (SignaGen Laboratories), FuGENE HD (Promega), or Lipofecta- mine (Invitrogen) according to the instructions provided by the manufacturers. Acknowledgments We gratefully acknowledge the expert help of Jonathan Boyd (Leica Microsystems) in performing STED microscopy. This paper is dedicated to the memory of our friend and colleague Susan L. Wearne. Author Contributions Conceived and designed the experiments: YK TK DSK. Performed the experiments: YK TK DSK. Analyzed the data: YK TK PD DSK. Contributed reagents/materials/analysis tools: PD. Wrote the paper: DSK. 6. Rasala BA, Ramos C, Harel A, Forbes DJ (2008) Capture of AT-rich chromatin by ELYS recruits POM121 and NDC1 to initiate nuclear pore assembly. Mol Biol Cell 19: 3982–3996. 7. Stavru F, Nautrup-Pedersen G, Cordes VC, Gorlich D (2006) Nuclear pore complex assembly and maintenance in POM121- and gp210-deficient cells. J Cell Biol 173: 477–483. 8. Stavru F, Hulsmann BB, Spang A, Hartmann E, Cordes VC, et al. (2006) NDC1: a crucial membrane-integral nucleoporin of metazoan nuclear pore complexes. J Cell Biol 173: 509–519. 9. Rayala HJ, Kendirgi F, Barry DM, Majerus PW, Wente SR (2004) The mRNA export factor human Gle1 interacts with the nuclear pore complex protein Nup155. Mol Cell Proteomics 3: 145–155. PLoS ONE | www.plosone.org 13 April 2012 | Volume 7 | Issue 4 | e36137