plasmids pbind, pact and pg5luc  (Promega)

Journal: Antiviral Research

Article Title: Application of a cell-based protease assay for testing inhibitors of picornavirus 3C proteases

doi: 10.1016/j.antiviral.2013.12.012

Figure Lengend Snippet: Principle and evaluation of the cell-based protease assay. (A) A protease expression construct is co-transfected with the pG5luc reporter plasmid into COS-1 cells. The protease construct expresses a chimeric protein which contains a GAL4 binding domain (GAL4BD) and a VP16 activation domain (VP16AD) between which part of the CVB3 polyprotein (15 C-terminal amino acids of 3A, 3B, 3C pro , and 15 N-terminal acids of 3D) is inserted. Active protease cleaves the chimeric protein at the 3C pro cleavage (arrow). If the protease is catalytically inactive, binding of GAL4BD to the GAL4 sequences in the reporter plasmid recruits VP16AD to the transcription start site, resulting in induction of FLuc expression. (B) The fusion protein is 3C pro -dependently cleaved. Plasmids pBind, pBind-VP16, pBind-3C pro (CVB3)-VP16, or pBind-3C pro [C147A](CVB3)-VP16 were co-transfected with pG5luc into COS-1 cells. The next day, cells were lysed and the proteins were separated by SDS–PAGE and stained with α-GAL4BD and α-Tubulin antibodies. (C + D) Inhibition of 3C pro activity results in induction of FLuc expression. COS-1 cells were co-transfected with protease constructs in combination with the pG5luc reporter and immediately treated with DMSO or AG7088 at 50 μM (C) or at the indicated concentrations (D). At 16 h post transfection, the cells were lysed and FLuc and RLuc were measured. Experiments were performed in triplicate and mean values ± SD are depicted.

Article Snippet: Plasmids pBind, pAct and pG5luc were derived from the CheckMateTM Mammalian Two-Hybrid System (Promega). pBind-VP16 was produced by ligating the VP16AD-coding sequence amplified from pAct into the XbaI and NotI sites of the multiple cloning site of pBind. pBind-VP16 was subsequently used for cloning all protease constructs using the SalI and MluI sites between GAL4BD and VP16AD.

Techniques: Protease Assay, Expressing, Construct, Transfection, Plasmid Preparation, Binding Assay, Activation Assay, SDS Page, Staining, Inhibition, Activity Assay

Journal: Antiviral Research

Article Title: Application of a cell-based protease assay for testing inhibitors of picornavirus 3C proteases

doi: 10.1016/j.antiviral.2013.12.012

Figure Lengend Snippet: Suitability of the CVB3 3C pro assay for high-throughput screening. (A) Determination of the Z ′ factor for the CVB3 3C pro assay. COS-1 cells were co-transfected with pBind-3C pro (CVB3)-VP16 and pG5luc, treated with 50 μM AG7088 and luciferase levels were measured the next day. The Gaussia curves displayed were fitted using nonlinear regression. (B) EGFP can also be used as a reporter for protease activity. COS-1 cells were co-transfected with pBind-3C pro (CVB3)-VP16 or the C147A mutant and pG5EGFP, treated with 50 μM AG7088 and EGFP was imaged after 2 days.

Article Snippet: Plasmids pBind, pAct and pG5luc were derived from the CheckMateTM Mammalian Two-Hybrid System (Promega). pBind-VP16 was produced by ligating the VP16AD-coding sequence amplified from pAct into the XbaI and NotI sites of the multiple cloning site of pBind. pBind-VP16 was subsequently used for cloning all protease constructs using the SalI and MluI sites between GAL4BD and VP16AD.

Techniques: High Throughput Screening Assay, Transfection, Luciferase, Activity Assay, Mutagenesis

Journal: Antiviral Research

Article Title: Application of a cell-based protease assay for testing inhibitors of picornavirus 3C proteases

doi: 10.1016/j.antiviral.2013.12.012

Figure Lengend Snippet: AG7088 and SG85 inhibit enterovirus 3C pro and FMDV 3C pro . (A) AG7088 and SG85 display activity against enterovirus 3C pro and FMDV 3C pro . Cells co-transfected with the constructs for the indicated proteases and pG5luc reporter were treated with 50 μM AG7088 or SG85 and the next day the luciferase levels were measured. Displayed are the FLuc levels. (B + C) AG7088 and SG85, but not AG7404 inhibit FMDV 3C pro , albeit with lower potency than CVB3 3C pro . Cells co-transfected with pG5luc and the CVB3 or FMDV 3C pro constructs were treated with a range of concentrations of AG7088 or AG7404 (B) or SG85 (C). The dashed lines represent the values obtained for the respective inactive mutants (upper dashed line) or the values obtained for the untreated wt constructs (lower dashed line). All experiments were performed in triplicate and values represent the mean FLuc ± SD.

Article Snippet: Plasmids pBind, pAct and pG5luc were derived from the CheckMateTM Mammalian Two-Hybrid System (Promega). pBind-VP16 was produced by ligating the VP16AD-coding sequence amplified from pAct into the XbaI and NotI sites of the multiple cloning site of pBind. pBind-VP16 was subsequently used for cloning all protease constructs using the SalI and MluI sites between GAL4BD and VP16AD.

Techniques: Activity Assay, Transfection, Construct, Luciferase

Journal: Biochemical and Biophysical Research Communications

Article Title: UAP56 is a novel interacting partner of Bcr in regulating vascular smooth muscle cell DNA synthesis

doi: 10.1016/j.bbrc.2012.03.022

Figure Lengend Snippet: (A) After over expressing Bcr and UAP56 in HeLa cells, total cell lysates (TL) were prepared and UAP56 and Bcr were immunoprecipitated using respective antibodies and Western blotting was performed with UAP56 antibody. (B) (Mammalian two-hybrid assay). HeLa cells were transfected with pBIND or pBIND-Bcr and pACT UAP56 full length or UAP56 fragments. Cells were harvested and luciferase assay performed. Full length UAP56 binds to Bcr as does UAP56 fragment 2 (amino acids 101-200). (**p<0.01). (C) HeLa cells were transfected with pBIND-Bcr and pACT UAP56 full length or UAP56 fragments as indicated (the pACT vector contains the herpes simplex virus VP16 activation domain). Bcr was immunoprecipitated with Bcr antibody and immunblot done with UAP56 antibody. Over expression of UAP56 fragment 2 [amino acids (aa) 101-200] blocked Bcr/UAP56 binding. Over expression of fragment 3 (aa 201-300) had no effect.

Article Snippet: Mammalian Two-hybrid assay HeLa cells were transfected in Opti-MEM (Invitrogen) with Lipofectamine mixture containing the pG5-luc vector and various pBIND and pACT plasmids (Promega) for 4 h. The pBIND vector contains the yeast GAL4–DNA-binding domain upstream of a multiple cloning region, and the pACT vector contains the herpes simplex virus VP16 activation domain upstream of a multiple cloning region.

Techniques: Expressing, Immunoprecipitation, Western Blot, Two Hybrid Assay, Transfection, Luciferase, Plasmid Preparation, Activation Assay, Over Expression, Binding Assay