(C) interphase cell showing a membrane-associated patch

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(C) interphase cell showing a membrane-associated patch. medial ring, and cortical actin patches (Marks and strains used in this study are listed in Table ?Table1.1. strains were grown in yeast extract (YE) medium or minimal medium with the appropriate supplements as described (Moreno strains GSK1059865 for two-hybrid analysis were grown on synthetic minimal medium with the appropriate supplements (Guthrie and Fink, 1991 ). Table 1 Strain list (1994) ?KGY1187h90and pBI-771and pBI-771This study ?KGY1974YPB2 carrying pBI-770 and pBI-771(Thornwood, NY) Axioskop microscope with the use of the appropriate set of filters. To visualize DNA and the cell wall, cells were stained with DAPI and Calcofluor as described (Balasubramanian genomic library (Barbet coding region was accomplished with the use of the Muta-Gene phagemid in vitro mutagenesis kit (cells (KGY28) were grown to midlog phase, harvested by vacuum filtration, transferred rapidly to a thin brass sample chamber, and then frozen in an HPM-010 high-pressure freezer (BalTech, Liechtenstein). The cells were then transferred under liquid nitrogen to a freeze substitution device in which their frozen water was dissolved GSK1059865 at ?90C with acetone containing 0.1% anhydrous glutaraldehyde, as described previously (Demeter (Deerfield, IL) Ultracut E microtome and picked up on Formvar-coated, carbon-stabilized slot grids. Grids with sections were incubated for 30 min in a blocking buffer containing PBS, 0.8% BSA, 0.1% fish skin gelatin, and 0.01% Tween 80. All antibodies were GSK1059865 diluted in this blocking buffer. All rinses between antibody applications used PBS containing 0.1% Tween 80. Affinity-purified rabbit anti-Arp2p antibodies (100 g/ml) were applied to the sections at a dilution of 1 1:20, incubated overnight at 4C, and then rinsed three times. Goat antibodies to rabbit IgG labeled with 10-nm colloidal gold (British Biocell International) were diluted 1:20 and applied to sections for 2 h at room temperature, followed by three rinses in the same buffer. A mouse mAb against actin (Amersham Pharmacia Biotech) diluted 1:200 was then applied to the sections for another overnight incubation at 4C. The sections were rinsed, and rabbit antibodies to mouse IgG labeled with 5-nm colloidal gold were applied to the sections for 2 h at room temperature. After rinsing, the specimens were fixed GSK1059865 with 0.5% glutaraldehyde in PBS, followed by staining with 2% uranyl acetate for 8 min and Reynolds lead citrate for 3 min. The sections were examined in a Phillips (Mahwah, NJ) CM10 electron microscope operating at 80 kV, and images were recorded on Kodak (Rochester, NY) 4489 film. Epitope Tagging A genomic copy of encoding GSK1059865 three copies of a hemagglutinin (HA) tag (3xHA-KAN cassette [Bahler genomic locus. Detection of HA-tagged Sop2p was accomplished by immunoblotting with the use of an anti-HA mAb (12CA5, Boehringer Mannheim, Indianapolis, IN), followed by a peroxidase-conjugated goat anti-mouse IgG (1:10,000; and genes were PCR amplified from either a cDNA library (Y2HL [James was amplified from cDNA with the primers 5-CTAACAAAACTGTCGACTAATAATGGACCCACATAATCC-3 and 5-GGATCAATAAAGTCGACATCTATCTTGGACC-3 and TaqPlus Precision DNA polymerase (Stratagene) in a PTC-100 programmable thermal controller (MJ Research) programmed as follows: 95C, 1 min; 65-1C per cycle, 1 min; 72C, 5 min (15 cycles); 95C, 1 min; 50C, 1 min; 72C, 10 min (30 cycles). The 0.5-kb fragment of the gene was amplified with the primers 5-CTCCCAGTATGTCGACAAATGCCTGCGTATCACTCG-3 and 5-CCTTGCACCGTCGACCTTTATAGAGATTTGTTC-3. Bait and prey plasmids were cotransformed into host strain YPB2 according to the method described previously (Gietz transcription as assayed by growth on medium lacking histidine was regarded as evidence of bait and prey protein interaction. Activation of GAL4 transcription was assayed by -galactosidase filter assays to confirm protein-protein interactions. GST Fusion Proteins The coding region was cloned into the vector pGEX-2T (Amersham Pharmacia Biotech) to produce a GST-Arc18p fusion protein. The fusion protein was produced in bacterial cells after induction with 0.4 mM isopropylthio–galactoside and purified from bacterial lysates according to the method described previously (Frangioni and Neel, 1993 ). The coding region of was cloned into pBluescript SK+ (Stratagene) and translated in vitro in the presence of [35S]methionine (35S-Trans label, ICN Pharmaceuticals) with the use of the TNT coupled reticulocyte lysate system (Promega, Madison, WI). Purified GST or GST-Arc18p bound to glutathione-agarose beads was mixed with 35S-labeled Arp2p in binding buffer (20 mM Tris-HCl, pH 7.0, 150 mM NaCl, 2 mM EDTA, 0.1% NP-40) and incubated at 4C for 1 h. The beads were then washed three times in binding buffer, and the proteins were resolved CBLL1 by SDS-PAGE, treated with Amplify (Amersham Pharmacia Biotech), and exposed to film. Photoaffinity Labeling The coding regions of thiamine-repressible promoter present in pREP41 (Basi Arp2 protein sequence is 44% identical (64% similar) to conventional actin but is 63C69% identical.