Journal:

Article Title: Xenopus small heat shock proteins, Hsp30C and Hsp30D, maintain heat- and chemically denatured luciferase in a folding-competent state

doi:

Figure Lengend Snippet: Luciferase (LUC) heat denatured in the presence of recombinant Hsp30C (30C) can be refolded in vivo after microinjection into Xenopus oocytes. LUC (0.2 μM) was incubated at 22°C (♦), or heat denatured alone at 42°C (X) or in the presence of either 6 μM bovine serum albumin (•) or 30C at 30C-LUC molar ratios of 1:1 (□), 10:1 (▴), or 30:1 (▵) for 15 minutes. Mixtures (containing 1.38 fmol of LUC in 26.7 nL) were microinjected into Xenopus oocytes, and LUC activity in the oocytes was monitored over time as described in “Materials and Methods.” Data are representative of 3–5 trials and shown as the mean ± SE

Article Snippet: The mixtures were then vortexed and diluted 10-fold into LUC reagent solution (Promega).

Techniques: Luciferase, Recombinant, In Vivo, Microinjection, Incubation, Activity Assay

Journal: Virology

Article Title: Dynamic association of gammaherpesvirus DNA with core histone during de novo lytic infection of primary cells

doi: 10.1016/j.virol.2011.09.024

Figure Lengend Snippet: Primary bone marrow macrophages were infected at an indicated MOI with wild type MHV68, and chromatin was harvested throughout a single cycle of replication and subjected to ChIP using anti-histone H3 or a negative control antibody (IgG), as indicated (A–D, F). Efficiency of immunoprecipitation of indicated MHV68 DNA sequences was normalized to the efficiency of immunoprecipitation of GAPDH (A) using the ΔΔCt method and is presented as relative enrichment over GAPDH (B–D, F). E. Total RNA was harvested at indicated times post infection and converted to cDNA in the presence or absence of reverse transcriptase (RT). Abundance of RTA sequences was measured by real time PCR and is presented as fold difference between the corresponding plus RT and minus RT samples. F. Primary macrophages were infected at an MOI of 1, chromatin harvested at indicated times, and subjected to ChIP using anti-histone H3 antibody. G, H. Viral DNA was measured in input samples by real time PCR at indicated time points post infection and normalized to corresponding GAPDH levels. For each panel, data were pooled from 2–5 independent experiments, with error bars representing one standard error of the mean.

Article Snippet: Precleared chromatin was diluted 10-fold in the immunoprecipitation buffer (0.01% SDS, 1.1% Triton-10X, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.1, and 176 mM NaCl, 10 μL Halt protease inhibitor cocktail [Thermo Scientific, Waltham, MA]), immunoprecipitated with 2 μg anti-histone H3 (AbCam, Cambridge, MA) or rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA) overnight, and incubated with 60 μL protein G for 2h at 4°C.

Techniques: Infection, Negative Control, Immunoprecipitation, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Virology

Article Title: Dynamic association of gammaherpesvirus DNA with core histone during de novo lytic infection of primary cells

doi: 10.1016/j.virol.2011.09.024

Figure Lengend Snippet: Primary murine embryonic fibroblasts (MEFs) were infected at an MOI of 1 with wild type MHV68, chromatin was harvested at the specified time points during a single round of viral replication, and subjected to ChIP using anti-histone H3 or a negative control antibody (IgG) as indicated (A–C). Efficiency of immunoprecipitation of indicated MHV68 DNA sequences was normalized to the efficiency of immunoprecipitation of GAPDH (A) using the ΔΔCt method and is presented as relative enrichment over GAPDH (B, C). D. Viral DNA was measured in input samples by real time PCR at indicated time points post infection and normalized to corresponding GAPDH levels. For each panel, data were pooled from 2–5 independent experiments, with error bars representing one standard error of the mean.

Article Snippet: Precleared chromatin was diluted 10-fold in the immunoprecipitation buffer (0.01% SDS, 1.1% Triton-10X, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.1, and 176 mM NaCl, 10 μL Halt protease inhibitor cocktail [Thermo Scientific, Waltham, MA]), immunoprecipitated with 2 μg anti-histone H3 (AbCam, Cambridge, MA) or rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA) overnight, and incubated with 60 μL protein G for 2h at 4°C.

Techniques: Infection, Negative Control, Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: Virology

Article Title: Dynamic association of gammaherpesvirus DNA with core histone during de novo lytic infection of primary cells

doi: 10.1016/j.virol.2011.09.024

Figure Lengend Snippet: Primary bone marrow macrophages were infected at MOI 10 with wild type MHV68 and treated with PBS or 1 g/ml of cidofovir for the duration of the experiment. Chromatin was harvested at 30 and 48h post infection and subjected to ChIP using anti-H3 (A, B). Efficiency of immunoprecipitation of indicated MHV68 DNA sequences was normalized to the efficiency of immunoprecipitation of GAPDH using ΔΔCt method. C. Viral DNA was measured in input samples by real time PCR at indicated time points post infection and normalized to corresponding GAPDH, with relative levels observed at 30h post infection in cidofovir treated cells set to 1. D. Relative number of MHV68 sequences associated with histone H3 under indicated conditions. This number was determined based on efficiency of immunoprecipitation with histone H3 antibody and relative viral DNA levels (calculated in C). For each panel, data were pooled from 2–3 independent experiments and error bars presented as one standard error of the mean.

Article Snippet: Precleared chromatin was diluted 10-fold in the immunoprecipitation buffer (0.01% SDS, 1.1% Triton-10X, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.1, and 176 mM NaCl, 10 μL Halt protease inhibitor cocktail [Thermo Scientific, Waltham, MA]), immunoprecipitated with 2 μg anti-histone H3 (AbCam, Cambridge, MA) or rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA) overnight, and incubated with 60 μL protein G for 2h at 4°C.

Techniques: Infection, Immunoprecipitation, Real-time Polymerase Chain Reaction