Journal: Physiological Genomics

Article Title: Superoxide scavenging and Akt inhibition in myocardium ameliorate pressure overload-induced NF-?B activation and cardiac hypertrophy

doi: 10.1152/physiolgenomics.00202.2009

Figure Lengend Snippet: TAB-induced NF-κB activation is attenuated by AdCu/ZnSOD and AdDNAkt. Representative images at day 5 (A) and summary data (B) show the effects of AdLacZ, AdCu/ZnSOD, or AdDNAkt on NF-κB-driven luciferase activity (photon emission) over time in TAB mice compared with sham-operated mice. NF-κB activation was measured by in vivo bioluminescent imaging using Xenogen IVIS-200. In the pseudocolored scale, areas of high photon emission are displayed as red and areas of low photon emission are displayed as blue. Data are means ± SE (n = 4/group) expressed relative to sham-operated mice. *P < 0.05 vs. sham surgery; †P < 0.05 vs. TAB-AdLacZ. DNAkt, dominant-negative form of Akt.

Article Snippet: EMSA was performed with 6 μg of nuclear protein incubated with a γ- 32 P-labeled NF-κB consensus oligonucleotide probe 5′-AGTTGAGGGGACTTTCCCAGGC-3′ (Promega, Madison, WI) as described earlier ( 52 ).

Techniques: Activation Assay, Luciferase, Activity Assay, In Vivo, Imaging, Dominant Negative Mutation

Journal:

Article Title: Nuclear Factor-?B Activation in Neonatal Mouse Lung Protects against Lipopolysaccharide-induced Inflammation

doi: 10.1164/rccm.200608-1162OC

Figure Lengend Snippet: Systemic LPS activates distinct nuclear factor (NF)-κB complexes in adult and neonatal mouse lungs. (A) Nuclear extracts from lung homogenates were incubated with radiolabeled oligonucleotides containing the NF-κB consensus sequence. Activated NF-κB complexes were present in the nuclear extracts of both LPS-treated neonatal and adult mice, beginning at 1 hour (lanes 3 and 11), peaking at 2 hours (lanes 4, 5, 12, 13) and decreasing by 4 hours (lanes 6 and 14). However, the primary complex in the neonates (lanes 4 and 5) was noted to have a slower speed of migration as compared with the primary complex found in the adults (lanes 12 and 13). Specificity of the bands was confirmed by the disappearance of the bands with the addition of 100-fold excess of cold oligonucleotide (Cold oligo) (lanes 7 and 15), and the reappearance of the bands with the addition of 100-fold excess of cold, mutated oligonucleotide (Mutant oligo) (lanes 8 and 16). Supershift analysis of the neonatal (B) and adult (C) NF-κB complexes present at 2 hours was performed by the addition of antibodies against the Rel family proteins: p65, p50, Rel B, and cRel. There is an upward shift of the neonatal NF-κB complex with the addition of antibodies against p50, and decreased intensity of the band with the addition of antibodies against p65. Supershift analysis of the adult complex demonstrates a shift in the position or intensity of the band only by the addition of p50 antibodies. Images are representative examples of n ⩾ 3 individual experiments.

Article Snippet: Electrophoretic Mobility Shift Assay Nuclear extracts obtained from whole mouse lung were used for DNA binding assays using γ- 32 P–labeled double-stranded oligonucleotides containing the NF-κB consensus sequence (Promega, Madison, WI) as previously described ( 12 ).

Techniques: Incubation, Sequencing, Migration, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Quadruplex formation by both G-rich and C-rich DNA strands of the C9orf72 (GGGGCC)8•(GGCCCC)8 repeat: effect of CpG methylation

doi: 10.1093/nar/gkv1008

Figure Lengend Snippet: Methylation type and salt influence electrophoretic migration patterns. Migration of [γ- 32 P] ATP end-labeled DNA (frozen and thawed at room temperature) in native 8% polyacrylamide gel. [γ- 32 P] ATP end-labeled (GGGGCC) 8 and (CCCCGG) 8 containing the indicated modifications were dissolved in 50 mM Tris–HCl (pH 7.4) with the indicated salt, incubated for 20 min and then electrophoresed for 2 h at 120 V (9 V/cm).

Article Snippet: Reactions were carried out in a 20-μl volume containing ∼80 fmol of [γ- 32 P]ATP end-labeled DNA with the indicated amounts of hnRNPK (Abnova).

Techniques: Methylation, Migration, Labeling, Incubation

Journal: Nucleic Acids Research

Article Title: Quadruplex formation by both G-rich and C-rich DNA strands of the C9orf72 (GGGGCC)8•(GGCCCC)8 repeat: effect of CpG methylation

doi: 10.1093/nar/gkv1008

Figure Lengend Snippet: Methylation type influences DNA-protein interactions. Human recombinant hnRNPK band-shift with the [γ- 32 P] ATP end-labeled d(GGGGCC) 8 or d(GGCCCC) 8 DNAs (∼80 fmol) and 0, 50, 200 or 400 ng of purified recombinant hnRNPK. DNAs are either not CpG methylated, or contain 5-methylcytosine or 5-hydroxymethylcytosine bases at each CpG within the sequence. Samples were pre-incubated with the protein in a buffer solution containing 10 mM Tris (pH 7.4), 50 mM KCl, 1 mM DTT, 2.5% glycerol, 5 mM MgCl 2 , and 0.05% Non-idet P40. Samples were electrophoresed on a 4% native polyacrylamide gel for 1 hour at 200 volts (15V/cm). Lanes were densimetrically assessed using ImageQuant software to quantify the percent bound (%).

Article Snippet: Reactions were carried out in a 20-μl volume containing ∼80 fmol of [γ- 32 P]ATP end-labeled DNA with the indicated amounts of hnRNPK (Abnova).

Techniques: Methylation, Recombinant, Electrophoretic Mobility Shift Assay, Labeling, Purification, Sequencing, Incubation, Software