β gal

Reporter assays using the luciferase (Luc) reporter gene under control of the IFN-β promoter (IFN-β pGL3; a gift from J. Hiscott) have been described previously (Scholle and Mason, 2005 (link); Wilson et al., 2008 (link)). Briefly, 150ng each of IFN-β pGL3 and pCMV β-Galactosidase (β-Gal) (Invitrogen) were co-transfected into HeLa cells, using the TransIT-LT1transfection reagent (Mirus). At 4h post-transfection, clarified NS1-containing supernatant or clarified control supernatant was added to the cells. For experiments using purified sNS1, the appropriate concentration of sNS1, or an equal volume of pCtrl, was prepared in full serum media. Heat-inactivation of sNS1 was achieved by heating the sample at 95°C for 1h. After 16h cells were either left untreated or were stimulated by adding 20 μg/ml poly(I:C) (pIC; Calbiochem) to the culture media and incubating for 4 hours. Following treatment, cells were lysed in reporter lysis buffer (0.1% TritonX-100, 10% glycerol, 2mM Dithiothreitol, 2mM trans 1,2 diaminocyclohexane-NNNN acetic acid, and 25mM Tris phosphate) and assayed for Luc and β-Gal activities using a Promega Luc assay system and an ONPG (o-nitrophenyl-β-D-galactopyranoside)-based β-Gal assay. β-Gal activity was used to normalize the Luciferase data for all experiments. All data are expressed as relative light units/mU of β-Gal activity.

To isolate β-gal and the associated proteins, yeast cells expressing Ub-P-gal or M-gal were grown in YP-galactose medium for 12 h for protein induction. Cells were collected and re-suspended in buffer (50 mM Tris-HCl, pH 7.3; 200 mM NaCl; 5 mM MgCl2; 1 mM EDTA; 1 mM PMSF; protease inhibitors) and then lysed by glass beads. Equal amounts of proteins (3–4 mg) were applied to a 1 ml column of p-aminobenzyl-1-thio-β-galactoside crosslinked to agarose beads at 4°C and the bound β-gal was eluted with 8% lactose. The presence of Ssa1p or β-gal in the eluent was determined by western-blot with antibodies against human Hsp70 (StressGen) or β-gal (Promega). Enzymatic activity of β-gal was assayed as described in ‘Yeast Protocol Handbook’ (Clonetech).