Gas2 Inhibits Cell Division CH domain alone form a single actin ring during wound healing. Bar, 10 mm. (T-W) Oocytes expressing GFP-Gas2 domain alone form a single Gas2 domain ring, which localizes between the double actin rings (arrows in U) during wound healing. The GFP-Gas2 domain does not co-localize with either actin ring. Bar, 10 mm. doi:10.1371/journal.pone.0024698.g004 html). Phylogenetic tree was also derived by aligning the different species’ amino acids with ClustalW. Gas2 domain information was acquired from http://pfam.sanger.ac.uk/protein?acc = P11862. Cloning DNA constructs were generated by PCR and cloned into pCS2- eGFP vector (a kind gift from Prof. Bill Bement, University of Wisconsin-Madison, USA). The full-length Gas2 (cDNA clone MGC:18565) was cloned with N-terminus GFP tag by using the forward primer 59-ATGTGCACTGCCCTGA-39 and reverse primer 59-TCATTTAATCTCCTTCTTAGCCTTG-39. Gas2 CH domain (with N-terminus GFP tag) was cloned with the forward primer 59-TGCACTGCCCTGAGCC-39 and reverse primer 59-TCATCCAGTACTCTTCTTTCCTGAAG-39; and its Gas2 domain (with N-terminus GFP tag) was cloned with the forward primer 59-AGGTATGGTGTGGAGCCTCCT-39 and reverse primer 59-TCATTTAATCTCCTTCTTAGCCTTGT- 39. All clones were sequenced to ensure the sequence correctness. Western blot GFP tagged Gas2 constructs were injected into Xenopus oocytes nuclei and oocytes were incubated for 48 hours to allow for relative protein expression. The oocytes were then sonicated in phosphate buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 2 mM KH2PO4 at pH 7.4) with ultrasound sonicator, and samples were centrifuged at 30,000 g for 10 minutes at 4uC. Supernatants were collected for Western blot analysis. Protein assays were performed using the BCA protein assay kit (Thermo). Protein samples were run on a 12% SDS- PAGE gel and transferred to 0.45 mm Trans-Blot nitrocellulose paper (Bio-Rad), which was blocked with blotto buffer (5% non-fat milk in PBS) after transferring. After washing in 0.1% Tween-20 (Fisher) in PBS (PBS-T), monoclonal mouse GFP antibodies (Roche, Clone 7.1 and 13.1) were used at 1:3000 dilution in blotto buffer incubated at room temperature for 2 hours. The non- specific antibody binding was washed 3 times with PBS-T. Primary antibodies were detected using goat anti-mouse IgG antibodies conjugated to horse radish peroxidase (HRP) (Molec- ular Probes) at 1:3000 dilution in PBS at room temperature for 1 hour, and followed by 3 times washing with PBS-T. Detection was performed using the ECL method. Protein expression and purification Gas2 (cDNA clone MGC:18565) was cloned into pTrcHis2B vector with 6X His tag at N-terminus using the forward primer 59- ATGTGCACTGCCCTGA-39 and reverse primer 59-TTTAA- TCTCCTTCTTAGCCTTG-39. Basically, transform pTrcHis- 2B-Gas2 construct into DH5a Escherichia coli to express full-length Gas2 protein. Protein was collected with Probond nickel-chelating resin (Invitrogen). The elution protein solution was kept in a dialysis bag (Spectra/Pov molecularporous membrane tubing, MWCO 12–14 kDa, Fisher 08-667B) in PBS at 4uC overnight to remove imidazole. The protein sample was re-concentrated with Microcon centrifugal filter devices (Millipore: YM-10), and then was aliquoted into small volume and frozen in liquid nitrogen. Samples were stored at 280uC for further use. The protein purity was tested by Coomassie-stained SDS-PAGE, and no significant additional bands were observed. Xenopus laevis eggs collection and fertilization Human chorionic gonadotropin (hCG) was injected into 3 adult Xenopus females (1000 U/ml, 0.5 ml per frog) to induce ovulation. Eggs were harvested in glass Petri dish by gently squeezing the female. Eggs were fertilized by adding up a piece of a testis in the Petri dish and swirling gently. Ninety minutes later, 40 ml of 2% cysteine (Sigma) at pH 8.0 was added for 5 minutes to de-jelly embryos. Cysteine was poured of and replaced with Marc’s modified ringers (MMR: 100 mM NaCl, 2 mM KCl, 1 mM MgCl2, 2 mM CaCl2 and 5 mM HEPES at pH 7.5). Embryos were incubated at 16uC. They develop more rapidly at room temperature. Microinjection Microinjection needles (10 ml Drummond microdispenser, Drummond Scientific Company) were pulled by Flaming/Brown micropipette puller (Model P-97, Sutter Instrument Company). Five nl of 12 mg/ml bovine serum albumin (BSA) in PBS solution or the same amount of purified full-length Gas2 protein in PBS solution was injected into one cell of 2-cell stage Xenopus embryos. Injections were performed using a Model PLI-100 Pico-Injector (Harvard Apparatus). Live imaging movies The movies were recorded at a rate of 1 frame per minute with Zeiss Axioskop 2 microscope. Zeiss AxioCam MRm color camera and Plan-Neofluar 2.5X/0.075 N.A. objective lens were used. The images were modified with Volocity version 5.3.0 software (PerkinElmer) and played at 10 frames per second in Apple QuickTime (*.mov) format. Microinjection experiments results statistics tabulation The post-injected embryos were examined for the arrested cell division phenotype under a stereo microscope. Statistics results were charted with Microsoft Office Excel software, and analysis was performed using Student’s t-test. Cyro sample preparation and immunofluorescence One hundred pg each of GFP, full-length GFP-Gas2, GFP-CH domain or GFP-Gas2 domain constructs were injected into one cell of 2-cell stage embryos cytoplasm. The injected embryos were incubated in MMR solution and developed until Stage 10 or 11 before cryo preparation. Embryos were fixed and embedded in 15%, then 25% fish gelatin (Norland). They were frozen on dry ice before cutting. Ten mm thickness of cryo sections were cut in 220uC cyrotome (Leica), and collected on Superfrost plus slides (Fisher). A polyclonal rabbit Gas2 antibody (against peptide 78–99 KLH conjugation, provided by Prof. Jacque Paiement at University of Montreal) at 1:300 dilution, or a polyclonal rabbit GFP antibody (Molecular Probes) at 1:200 dilution, and a monoclonal alpha tubulin antibody (Sigma, DM1A) at 1:200 dilution were used. A goat-anti rabbit antibodies coupled to Alexa Fluor 488 (Molecular Probes) at 1:200 dilution, and a goat-anti mouse antibodies coupled to Alexa Fluor 546 (Molecular Probes) at 1:200 dilution were used for the staining. Nuclei were stained with DAPI (Molecular Probes) (0.5 g/ml) in PBS for 10 minutes. The yolk auto-fluorescence was quenched with 0.2% Eriochrome black (Sigma) in PBS for 10 minutes. Finally, the slides were PLoS ONE | www.plosone.org 9 September 2011 | Volume 6 | Issue 9 | e24698