To generate S2 cell expression constructs, PCR fragments encoding GroWT, GroDGP, GroDCcN, GroDSP, and GroDCR with an N-terminal FLAG tag (DYKDDDDK) and containing 59 XhoI and 39 SpeI restriction sites were inserted into the S2 cell expression acceptor vector pMK33-BD [57]. A Gro genomic rescue construct bearing a 10,000 bp gro- containing fragment from the right arm of chromosome 3 (spanning genomic coordinates 21,866,400 to 21,876,400) was created using the attP-attB-P[acman] recombineering system [42]. The left and right homology arms were amplified with primers sets 1 and 2 (Table S1) using BAC13F13 (Chori) as the DNA template and then inserted into the transformation vector attB-P(acman). SW102 cells containing BAC13F13 were then transformed with linearized attB-P(acman) containing the homology arms. Accurate gap repair was verified with primer sets 3 and 4 (Table S1) and the correctly recombined plasmid (named attB-P(acman)-gro 10 kb). To generate genomic rescue constructs encoding the Gro central domain deletions GroDGP, GroDCcN, GroDSP, we used attB- P(acman)-gro 10 kb as a PCR template. Primers used to generate these rescue constructs are given in Table S1. PCR amplification products encoding the genomic regions to the left and right of the site of the deletions were inserted into attB-P(acman). All rescue constructs were introduced into flies containing an attP docking site located at 2L-22A by phiC31 site-specific transgenesis (Rainbow Transgenic Flies, Inc., fly stock 9752 (22A)). Disorder prediction algorithms Disorder probability in Gro was predicted using the PONDR- FITTM and FoldIndex algorithms [40,41]. These algorithms take advantage of the discovery that intrinsically disordered proteins have significantly different amino acid sequences than do ordered proteins. Specifically, disordered proteins display low sequence complexity, a low content of bulky hydrophobic amino acids, and a high proportion of charged and polar amino acids. The algorithms were developed using databases of known disordered and ordered proteins to train artificial neural networks to assign protein disorder scores to moving windows of amino acids across proteins [39]. For the PONDR-FITTM algorithm, regions displaying scores consistently less than 0.5 are likely to be ordered, while regions displaying scores consistently greater than 0.5 are likely to be disordered. For the FoldIndex algorithm, regions displaying scores consistently below 0 are likely to be disordered, while regions displaying scores consistently above 0 are likely to be ordered. Gro genomic rescue experiments The progeny of flies containing one of two lethal gro mutant alleles, groMB12 (a strong hypomorph) or groMB36 (a null) [16], balanced over TM3 and a rescue construct balanced over CyO were examined to determine the fraction that were homozygous for the gro mutant allele. The expected ratio for 100% rescue is one-third gro/gro to two-thirds gro/TM3 progeny. Embryo and wing disc immunoblots To examine Gro expression in embryo, 50 embryos produced by the female progeny of a pUASP-Gro6Mat-Gal4 cross were placed in 40 ul of SDS-PAGE sample buffer (60 mM Tris-Cl [pH 6.8], 2% SDS, 10% glycerol, 5% b-mercaptoethanol, and 0.01% bromophenol blue), mashed, and boiled. Samples were analyzed by 8% SDS-PAGE and probed with either a 1:500 dilution of mouse anti-Gro monoclonal antibody (Hybridoma Bank) or a 1:10,000 dilution of mouse anti-tubulin monoclonal antibody (Sigma) using the Millipore dry blot method. Blots were subsequently incubated with a 1:10,000 dilution of secondary Groucho Central Domains Instruct Development antibody conjugated to horseradish peroxidase (CalBiochem) and signal was detected by enhanced chemiluminescence (ECL) with SuperSignal West Pico substrates (Pierce). To examine Gro expression in wing discs, forty third instar wing imaginal discs from the progeny of a pUASP-Gro6Ser-Gal4 cross were placed into 30 ul of SDS-PAGE sample buffer. Samples were processed and analyzed as described for the embryo immunoblots. Coimmobilization assays with His-tagged Gro variants pET17b-Gro [17] was used to express wildtype full-length Gro, and pET17b-GroDGP, pET17b-GroDCcN, pET17b-GroDSP, pET17b-GroDCR were used to express the Gro central domain deletion variants. pET3c-His-tagged N-terminal Gro (2–194) wild- type, Gro (2–194) 40/89, and Gro (2–194) 38/87 [18] were used to express wild-type and point mutants of GroN fused to a 6xHis- tag. For cotranslation, constructs encoding two forms of Gro were mixed before being added to the TNT T7 quick-coupled transcription-translation system (Promega) in the presence of [35S]-methionine. 10% of the translation product was reserved for analysis of the input, while the remaining 90% was diluted into binding buffer (25 mM HEPES [pH 7.6], 450 mM NaCl, 10 mM imidazole, 0.1% Tween 20, 1 mM dithiothreitol) and incubated with Nickel-Nitrilotriacetate (Ni-NTA) beads (QIAGEN) over- night at 4uC. Beads were then washed extensively with binding buffer. Proteins bound to the beads were eluted with SDS-PAGE sample buffer. Samples and reserved input material were analyzed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. GST-pulldown assays GST-histone tail fusion proteins were expressed in bacteria and purified as described previously using plasmids pGEX-2T-H3, pGEX-2T-H4, and pGEX-2T-control [58]. Gro deletion variants were translated in vitro from the pET17b constructs in the presence of [35S]-methionine using the TNT T7 coupled reticulocyte lysate system (Promega). In vitro translated Gro variants were incubated overnight with GST fusion proteins immobilized on glutathione beads at 4uC in HEMNK buffer (40 mM HEPES at pH 7.5, 5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, 0.5% NP-40, 0.1 M KCl). Following binding, the beads were washed extensively with HEMNK buffer. Proteins were eluted in SDS-PAGE sample buffer and the eluates, as well as the 10% reserved input material, were resolved by 8% SDS-PAGE and visualized by autoradiography. GST-Brk fusion proteins were expressed with pGEX-5x-1-Brk(441–589) [18] and pGEX-5xl- control vectors as described for the GST-histone tail fusions, except that the cells were lysed by two passes through a microfluiditor in STE buffer (0.15 M NaCl, 10 mM Tris-Cl, pH 8, 1 mM EDTA) with 5 mM DTT, 1% Triton-X 100 and protease inhibitors. Lysed cells were cleared by centrifugation. GST-pulldown assays to examine binding to vitro translated Gro deletion variants were performed as described for the GST-histone tail pulldown experiments, except the binding buffer was PBS containing 340 mM NaCl, 1 mM EDTA, 0.1% NP-40, and 10% BSA and the washes were with PBS containing 290 mM NaCl. Proteins were eluted in SDS-PAGE sample buffer and the eluate and 2% reserved input material were resolved by 8% SDS-PAGE and visualized by autoradiography. Preparation of embryonic cuticles Mat-Gal4 virgin females were crossed with transgenic males containing UASp constructs encoding Gro deletion variants or with control w1118 males and incubated at 25uC. Embryos were collected from F1 females for 3 hours and placed at 25uC for PLoS ONE | www.plosone.org 13 February 2012 | Volume 7 | Issue 2 | e30610