phosphonf-kb elisa  (Cell Signaling Technology Inc)

Journal: Immunity

Article Title: C-type lectin DC-SIGN modulates Toll-like receptor signaling via Raf-1 kinase-dependent acetylation of transcription factor NF-kappaB.

doi: 10.1016/j.immuni.2007.03.012

Figure Lengend Snippet: Figure 5. DC-SIGN-Mediated Raf-1-Dependent Signaling Induces p65 Phosphorylation on Ser276 and Acetylation, after Concur- rent TLR Signaling (A) DC-SIGN signaling does not influence nuclear translocation of different NF-kB subunits. DNA-binding ELISA of the NF-kB subunits p65, RelB, c-Rel, p50, and p52 in nuclear extracts of human iDCs treated as indicated for 1 hr. NF-kB was allowed to bind to oligonucleotides containing the NF-kB-binding consensus sequence; specific antibodies were used to detect the different subunits within the bound complexes. Results are pre- sented as the means ± SD from two independent experiments. (B and C) DC-SIGN signaling induces p65 phosphorylation on Ser276 through Raf-1. ELISA of phospho-Ser536-p65 (B) or phospho-Ser276-p65 (C) in cell lysates of human iDCs treated as indicated for 30 min; cells were preincubated for 2 hr with Raf inhibitor GW5074 as indicated. Results are pre- sented as the means ± SD from three independent experiments. (D) DC-SIGN induces acetylation of p65. ELISA with acetyl-lysine antibodies on captured p65 from cell lysates of human iDCs treated as indicated for 30 min; cells were preincubated with Raf inhibitor GW5074 as in (C). Results are presented as the means ± SD from three independent experiments. (E) Acetylation of p65 is essential to ManLAM-induced IL-10 upregulation. Relative IL-10 mRNA production by human iDCs treated with different ligands for 6 hr as indicated; cells were preincubated for 2 hr with anacardic acid (AA), an inhibitor of the histone acetyltransferases p300 and CBP. IL-10 mRNA was determined as described in Figure 1A. Results are presented as the means ± SD from three independent experiments.

Article Snippet: DNA Binding, Acetylation, and Phosphorylation of NF-kB DC nuclear extracts were prepared with the NucBuster protein extraction kit (Novagen, Madison, WI), and 5 mg of nuclear extract was used to determine the specific subunits within the DNA binding NF-kB dimers with the TransAM NF-kB family kit (Active Motif, Carlsbad, CA), according to the manufacturer’s protocol. p65 from cell lysates was captured with the Pathscan phosphoNF-kB p65 (Ser536) sandwich ELISA kit (Cell Signaling), according to the manufacturer’s protocol.

Techniques: Phospho-proteomics, Translocation Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Sequencing

Journal: Immunity

Article Title: C-type lectin DC-SIGN modulates Toll-like receptor signaling via Raf-1 kinase-dependent acetylation of transcription factor NF-kappaB.

doi: 10.1016/j.immuni.2007.03.012

Figure Lengend Snippet: Figure 6. DC-SIGN-Induced Acetylation Prolongs Nuclear p65 DNA Binding and Enhances the IL-10 Transcription Rate (A) DC-SIGN signaling prolongs nuclear p65 DNA binding compared to LPS signaling alone. DNA binding was determined as described in Figure 5A. Results are presented as the means ± SD from two independent experiments. (B) Acetylation of p65 prolongs nuclear p65 DNA binding. ELISA of p65 in nuclear extracts of hu- man iDCs treated similar as in (A); cells were pre- incubated with Raf inhibitor GW5074 and HAT inhibitor anacardic acid (AA) as in Figures 5C and 5E. DNA binding was determined as in (A). Results are presented as the means ± SD from two independent experiments. (C) DC-SIGN signaling enhances the IL10 tran- scription rate. Relative IL-10 transcription rate in nuclei isolated from human iDCs treated sim- ilar as in (A); cells were preincubated for 2 hr with the blocking DC-SIGN antibody AZN-D2 as indi- cated. Transcription by isolated nuclei was initi- ated in the presence of 16-biotin-UTP; after 20 min of transcription, nuclei were lysed, RNA with incorporated biotin was isolated, and quan- titative real-time PCR analysis was performed to determine the relative IL-10 RNA production. The amount of RNA produced in 20 min is repre- sentative of the transcription rate. The IL-10 RNA production by LPS-stimulated cells was set at 1. Results are presented as the means ± SD from two independent experiments.

Article Snippet: DNA Binding, Acetylation, and Phosphorylation of NF-kB DC nuclear extracts were prepared with the NucBuster protein extraction kit (Novagen, Madison, WI), and 5 mg of nuclear extract was used to determine the specific subunits within the DNA binding NF-kB dimers with the TransAM NF-kB family kit (Active Motif, Carlsbad, CA), according to the manufacturer’s protocol. p65 from cell lysates was captured with the Pathscan phosphoNF-kB p65 (Ser536) sandwich ELISA kit (Cell Signaling), according to the manufacturer’s protocol.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation, Isolation, Blocking Assay, Real-time Polymerase Chain Reaction, Produced