Journal: Oncotarget

Article Title: Decreased severity of collagen antibody and lipopolysaccharide-induced arthritis in human IL-32β overexpressed transgenic mice

doi:

Figure Lengend Snippet: A. CAIA with LPS treated mice joint tissues (two animals) were lysated and detected with antibodies against p50, p65, Histion H1, p-STAT3, and STAT3 using western-blotting. The DNA-binding activity of NF-κB and STAT3 was detected by EMSA B. IKK, p-IKK, IκB, p-IκB, MMP-3, MMP-9, iNOS, and COX-2 were detected using specific antibodies. The experiments shown in Fig. were repeated with another three set of two animals. The results were similar.

Article Snippet: The membrane was incubated with specific antibodies: mouse monoclonal antibodies against MMP9, p-p65, p65, p-IκB, STAT3, p-STAT3, histone H1 and β-actin (1:500 dilution, Santa Cruz Biotechnology Inc. Santa Cruz, CA, USA), Goat rabbit polyclonal MMP3 (1:500 dilution, Santa Cruz Biotechnology Inc. Santa Cruz, CA, USA), rabbit polyclonal for p50, p-IKKα, IKKα, p-IKKβ, IKKβ, p-IκB and IκB (1:500 dilution, Santa Cruz Biotechnology Inc.) and iNOS and COX-2 (1:1000 dilution, Cayman Chemical, Ann Arbor, Mich, USA).

Techniques: Western Blot, Binding Assay, Activity Assay

Journal: Cancer Research

Article Title: Noscapine, a Benzylisoquinoline Alkaloid, Sensitizes Leukemic Cells to Chemotherapeutic Agents and Cytokines by Modulating the NF-κB Signaling Pathway

doi: 10.1158/0008-5472.can-09-4230

Figure Lengend Snippet: Figure 5. Noscapine inhibits TNF-dependent IκBα phosphorylation, IκBα degradation, p65 phosphorylation, and p65 nuclear translocation. A, noscapine inhibits TNF-induced activation of NF-κB. KBM-5 cells were incubated with 50 μmol/L noscapine for 12 h, treated with 0.1 nmol/L TNF for the indicated times, and analyzed for NF-κB activation by EMSA. B, left, effect of noscapine on TNF-induced IκBα degradation and phosphorylation. Cells were incubated with 50 μmol/L noscapine for 12 h and treated with 0.1 nmol/L TNF for the indicated times. Cytoplasmic extracts were prepared, fractionated on SDS-PAGE, and electrotransferred to nitrocellulose membrane. Western blot analysis was performed using the indicated antibody. Anti–β-actin antibody was used as a loading control. Right, effect of noscapine on phosphorylation of IκBα by TNF. Cells were preincubated with 50 μmol/L noscapine for 12 h, incubated with 50 μg/mL ALLN for 30 min, and treated with 0.1 nmol/L TNF for 10 min. Cytoplasmic extracts were fractionated and then subjected to Western blot analysis using antibody to phosphorylated IκBα. The same membrane was reblotted with antibody to IκBα. C, left, noscapine inhibits TNF-induced IκBα kinase activity. Whole-cell extracts were immunoprecipitated with antibody against IKKα and analyzed by an immune complex kinase assay. To examine the effect of noscapine on expression of IKK protein, whole-cell extracts were fractionated on SDS-PAGE and examined by Western blot analysis using antibodies to IKKα and IKKβ. Right, direct effect of noscapine on IKK activation induced by TNF. Whole-cell extracts were prepared from KBM-5 cells treated with 1 nmol/L TNF and immunoprecipitated with antibody to IKKα. The immune complex kinase assay was performed with the indicated concentrations of noscapine. D, effect of noscapine on TNF-induced p65 nuclear translocation and phosphorylation. Cells were incubated with 50 μmol/L noscapine for 12 h and treated with 0.1 nmol/L TNF for the indicated times. Nuclear extracts were prepared and performed for Western blot analysis using the indicated antibody. Anti-PARP was used as a loading control for nuclear extract.

Article Snippet: Antibodies to IKKα, IKKβ, and cellular caspase-8 (FLICE)–like inhibitory protein (cFLIP) were kindly provided by Imgenex.

Techniques: Phospho-proteomics, Translocation Assay, Activation Assay, Incubation, SDS Page, Membrane, Western Blot, Control, Activity Assay, Immunoprecipitation, Immune Complex Kinase Assay, Expressing