Journal: Journal of Biological Chemistry

Article Title: Isoform Specificity of Ankyrin-B

doi: 10.1074/jbc.m506697200

Figure Lengend Snippet: FIGURE 1. Identification of an inter-domain interaction within ankyrin-B. A, domain organization of 220-kDa ankyrin-B. Ankyrin-B contains a membrane-binding domain, spectrin-binding domain, death domain, and C-terminal domain. The combination of death and C-terminal domain is termed the “regulatory domain.” B, ankyrin-B regulatory domain interacts with the membrane-binding domain in yeast two-hybrid assays. The ankyrin-B regulatory domain (fused with DNA-binding domain pAS2-1; Bait) was co- transformed into AH109 yeast strain with various ankyrin-B Prey constructs representing various ankyrin-B domains (fused to the DNA activation domain, pACT2). Positive inter- action was assessed by growth on media lacking adenine, histidine, leucine, tryptophan (-AHLT). We observed a positive interaction only when pAS2-1 regulatory domain was expressed with pACT2 membrane-binding domain.

Article Snippet: Fragments were PCR-amplified and inserted either in the pACT2 (GAL-4 activation domain vector) or pAS2-1 (GAL-4 DNA-binding domain vector).

Techniques: Membrane, Binding Assay, Transformation Assay, Construct, Activation Assay

Journal: Journal of Biological Chemistry

Article Title: Isoform Specificity of Ankyrin-B

doi: 10.1074/jbc.m506697200

Figure Lengend Snippet: FIGURE 3. Identification of the ankyrin-B membrane-binding domain site on the ankyrin-B regulatory domain. Yeast AH109 cells were co-transformed with ankyrin-B membrane-binding domain (fused with GAL-4 DNA activation domain) and one of ten Prey plasmids containing full-length or partial sequence of the ankyrin-B regulatory domain (amino acids 1445–1840). The death domain does not bind the membrane- binding domain while the C-terminal domain alone maintains binding affinity similar to the regulatory domain construct. The minimal binding region within the C-terminal domain is between amino acids 1556 and 1630. Deletion of this minimal binding region, D7, eliminates the intramolecular interaction. FIGURE 4. Identification of amino acids within the ankyrin-B C-terminal domain required for ankyrin-B inter-domain interaction. A, amino acid sequence within the C-terminal domain that contains membrane-binding domain activity. Amino acids cho- sen for alanine conversion are highlighted in red. Alanine-scanning mutants were gen- erated in the regulatory domain (pAS2-1) construct (see “Material and Methods” for details). B, a total of nine alanine-scanning mutants were screened for loss of binding to themembrane-bindingdomain(pACT2)revealingthataminoacidsGlu1597,Glu1598,and Asp1599 (regulatory 1597EEDAAA (pAS2-1)) are required for binding the membrane-bind- ing domain. The remaining eight mutants showed equivalent binding to the non-mu- tated regulatory domain.

Article Snippet: Fragments were PCR-amplified and inserted either in the pACT2 (GAL-4 activation domain vector) or pAS2-1 (GAL-4 DNA-binding domain vector).

Techniques: Membrane, Binding Assay, Transformation Assay, Activation Assay, Sequencing, Construct, Activity Assay

Journal: PLoS Pathogens

Article Title: A SAP30 Complex Inhibits IFN-β Expression in Rift Valley Fever Virus Infected Cells

doi: 10.1371/journal.ppat.0040013

Figure Lengend Snippet: (A) L929 wt330 cells, carrying an integrated wild type muIFN-β promoter fused to CAT reporter gene, were mock infected or infected by RVFV ZH or C13 or with NDV. Total cell extracts were prepared at 4, 6 and 8 h p.i. and CAT actvity was measured. (B) Non-confocal conventional fluorescence microscopy was used to analyze the nuclear distribution of NSs filaments in murine L929 cells infected by C13 or ZH. Presence of NSs filament detected using rabbit polyclonal anti-NSs antibody (green) or total DNA distribution revealed with Hoechst 33258 are shown respectively, in left and middle panels. Merged images are shown in right panels. Scale bars, 10 μm. (C) For yeast two-hybrid screening, AH109 yeast were co-transformed by pACT2-SAP30 1–152 that expressed Gal4 transactivating domain fused to the open reading frames corresponding to the 152 first aa of SAP30 and pGBKT7, pGBKT7-NSs ZH , pGBKT7-NSs C13 , pGBKT7-NSs TOS , or pGBKT7-NSs GER in which NSs from RVFV ZH or C13 or NSs proteins from Toscana (TOSV) and Germiston (GERV) bunyaviruses were fused to the Gal4 DNA-binding domain. The values of β galactosidase activity represent at least four independent experiments with SD bars. (D) Two-hybrid system using full length wild type NSs ZH or the deleted forms. The numbers indicate the amino acid position in the reference sequence. The sequence lacking amino acids 16–198 correspond to C13. (E) GST-NSs (left panel) or GST-SAP30 (right panel) was incubated with an extract from 293 cells transfected with the HA tagged-SAP30 expressing plasmid (left panel) or from ZH infected L929 cells (right panel). After extensive washing, the proteins bound to the beads were analysed by western blots using antibodies against HA (left panel) or NSs (right panel). The Coomassie blue staining showing that equivalent amounts of GST fusion proteins were loaded on the beads is not shown. (F) HEK 293 cells were transfected with either pCS2-Myc (lanes 1,2) or pCS2-Myc-SAP30 (lane 3) and either not infected (lane 1) or infected with ZH (lanes 2,3). Cell lysates were precipitated with anti-myc (9E10) antibody. Crude lysates (input) and the precipitated proteins (IP) were detected with anti-myc and anti-NSs antibodies.

Article Snippet: The plasmid pCi-HA-SAP30 was constructed by inserting the HA-tagged full length murine SAP30 cassette from the pACT2 plasmid into the pCi vector at the BglII site (Promega).

Techniques: Infection, Fluorescence, Microscopy, Two Hybrid Screening, Transformation Assay, Binding Assay, Activity Assay, Sequencing, Incubation, Transfection, Expressing, Plasmid Preparation, Western Blot, Staining