Journal: The Journal of Biological Chemistry

Article Title: Interleukin-20 Promotes Migration of Bladder Cancer Cells through Extracellular Signal-regulated Kinase (ERK)-mediated MMP-9 Protein Expression Leading to Nuclear Factor (NF-κB) Activation by Inducing the Up-regulation of p21 WAF1 Protein Expression *

doi: 10.1074/jbc.M112.410233

Figure Lengend Snippet: IL-20-induced activation of IKK, phosphorylation and degradation of IκBα, and translocation of p65 subunits in bladder cancer cells. A, cells were treated with IL-20 (50 ng/ml) for 24 h and then incubated with the indicated antibody for 30 min. They were assayed by EMSA. Preimmune serum (PIS) was included as the negative control. B, IL-20-induced phosphorylation and degradation of IκBα. The cells were incubated with IL-20 (50 ng/ml) for the indicated times. Cytoplasmic extracts were isolated and subjected to immunoblot using antibodies against IκBα or phospho-IκBα. Tubulin was used as a loading control protein. C, activation of IKK by IL-20. The cells were incubated with IL-20 (50 ng/ml) for differing time periods. Whole cell extracts were immunoprecipitated with antibody against IKK-α/β and subjected to immune complex kinase assay. To examine the effect of IL-20 on the level of expression of IKK proteins, whole cell extracts were examined by immunoblot using anti-IKK-α and anti-IKK-β antibodies. D and E, IL-20 induced the nuclear translocation of p65. The cells were either untreated or pretreated with IL-20 (50 ng/ml) for the indicated times. Cytoplasmic (C) and nuclear extracts (N) were prepared and analyzed by immunoblot using antibodies against p65 or phospho-p65 (p-p65). For loading control of nuclear and cytoplasmic protein, anti-tubulin and lamin B antibodies were used, respectively. F, ChIP analysis of the MMP-9 NF-κB-binding site. The cells were treated with IL-20 for the indicated time intervals. DNA immunoprecipitated by an anti-NF-κB p65 antibody was purified. Immunoprecipitated (IP) DNA was amplified by PCR using primers. The input represents PCR products from chromatin pellets prior to immunoprecipitation.

Article Snippet: Anti-IKK-α and anti-IKK-β antibodies were obtained from Imgenex (San Diego).

Techniques: Activation Assay, Translocation Assay, Incubation, Negative Control, Isolation, Western Blot, Immunoprecipitation, Immune Complex Kinase Assay, Expressing, Binding Assay, Purification, Amplification

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-20 Promotes Migration of Bladder Cancer Cells through Extracellular Signal-regulated Kinase (ERK)-mediated MMP-9 Protein Expression Leading to Nuclear Factor (NF-κB) Activation by Inducing the Up-regulation of p21 WAF1 Protein Expression *

doi: 10.1074/jbc.M112.410233

Figure Lengend Snippet: ERK1/2 inhibitor U0126 suppressed the IL-20-induced activation of IKK, phosphorylation and degradation of IκBα, and translocation of p65 subunits in bladder cancer cells. A, C, and D, U0126 inhibited the phosphorylation and degradation of IκBα and translocation of p65 subunit in cells. The cells were untreated or treated with U0126 (10 μm) for 4 h and then stimulated with IL-20 (50 ng/ml) for 15 min. Cytoplasmic (C) and nuclear (N) extracts were prepared and analyzed by immunoblot using antibodies against IκBα, phospho-IκBα, p65, or phospho-p65 (p-p65). For loading control of nuclear and cytoplasmic protein, antibodies specific for tubulin and lamin B were used, respectively. B, U0126 inhibits IL-20-induced IKK activity. The cells were untreated or treated with U0126 (10 μm) for 4 h, followed by treatment with IL-20 for 15 min. The IKK proteins were then immunoprecipitated with anti-IKK-α/β antibody. The levels of activation of GST-IkBα were detected by IKK assay. The immunoprecipitated IKK protein levels were examined from whole cell extracts by immunoblot using anti-IKK-α and anti-IKK-β antibodies.

Article Snippet: Anti-IKK-α and anti-IKK-β antibodies were obtained from Imgenex (San Diego).

Techniques: Activation Assay, Translocation Assay, Western Blot, Activity Assay, Immunoprecipitation

Journal: Scientific Reports

Article Title: A phenotypic high-content, high-throughput screen identifies inhibitors of NLRP3 inflammasome activation

doi: 10.1038/s41598-021-94850-w

Figure Lengend Snippet: NLRP3 inflammasome inhibitor hits validation in the speck assay. ( a ) Schematic of the priming protocol. ( b ) Schematic of the activation protocol. ASC-mCherry iBMDM cells were treated with indicated concentrations of ( c ) PU H71, ( d ) MPC-3100, ( e ) momelotinib, ( f ) CEP-33779, ( g ) ACHP or ( h ) MLN120B with LPS (1 µg/ml) for 2 h followed by nigericin treatment (10 µM, 2 h) after which PFA was added (priming—blue trace) and specks counted. In a parallel experiment the compounds were added after LPS treatment just prior to nigericin (activation—red trace). The images are representative of the nuclei and speck formation for each compound at 10 µM in both priming and activation protocols. Data are presented as mean ± SEM, n = 3 independent experiments. Data in ( c – h ) was analyzed using GraphPad Prism version 7 software ( https://www.graphpad.com/scientific-software/prism/ ).

Article Snippet: MCC950 (CAS number: 256373-96-3) was purchased from Tocris; PU H71 (Cat. number: 1856) and momelotinib (CAS number: 1056634-68-4) were purchased from Axon Medchem; MPC-3100 (Cat. number: A4063) was purchased from ApexBio; MLN120B (CAS number: 783348-36-7), CEP-33779 (CAS number: 958025-66-6) and ACHP (CAS number: 406209-26-5) were purchased from MedChemExpress.

Techniques: Activation Assay, Software