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: Journal of Cellular Physiology

Article Title: Galectin‐3 exacerbates ox‐LDL‐mediated endothelial injury by inducing inflammation via integrin β1‐RhoA‐JNK signaling activation

doi: 10.1002/jcp.27910

Figure Lengend Snippet: Gal‐3 promotes ox‐LDL‐induced inflammatory responses via NF‐κB activation and enhances the expression of related inflammatory factors, chemokines, and adhesion molecules. After exposure to Gal‐3, ox‐LDL or their combination, the total and phosphorylated expression of p65, IKKα, and IKKβ was examined by WB demonstrated by histogram (a and b); the levels of IL‐6, IL‐8, and IL‐1β and chemokines (CXCL‐1 and CCL‐2) were measured by ELISA. (c and d) The expression of VCAM‐1 and ICAM‐1 was detected by WB and the relative protein expression was given by histogram. (c) Each experiment was performed in triplicate. * p < 0.05, ** p < 0.01, ## p < 0.01

Article Snippet: Subsequently, the membranes were incubated with the following primary antibodies at 4°C overnight: integrin β1 (1:1000, cat.24693, abcam), RhoA (1:1000, cat.6352, Affinity), phosphorylation of RhoA (GTP‐RhoA) (1:500, cat.211164, abcam), JNK (1:1000, cat. AF6319, Affinity), p‐JNK (1:1000, cat. AF3320, Affinity), ICAM‐1 (1:1000, cat. DF7413, Affinity) and VCAM‐1 (1:1000, cat.DF6082, Affinity), NF‐κB P65(1:1000, AF5006, Affinity), PhosphoNFκB P65(1:1000, AF2006, Affinity), IKKα (1:1000, AF6012, Affinity), IKKβ (1:1000, bs‐4880R, Bioss, China), Phospho‐IKKβ(1:1000, bs‐3232R, Bioss, China).

Techniques: Activation Assay, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cellular Physiology

Article Title: Galectin‐3 exacerbates ox‐LDL‐mediated endothelial injury by inducing inflammation via integrin β1‐RhoA‐JNK signaling activation

doi: 10.1002/jcp.27910

Figure Lengend Snippet: Gal‐3 induced expression of inflammatory factors promoted HUVECs injury via integrin β1‐RhoA‐JNK pathway. HUVECs, exposing to the ox‐LDL alone or in combination with Gal‐3 were further treated with integrin β1‐siRNA or JNK inhibitor (SP600125). The total and phosphorylated expression of p65,IKKα and IKKβ after the above treatment was examined by WB and demonstrated by histogram. (a and b).The levels of inflammatory cytokines and chemokines were then measured by ELISA. (c and d) The expression of adhesion molecules (ICAM‐1 and VCAM‐1) was detected by WB (e). * p < 0.05, ** p < 0.01

Article Snippet: Subsequently, the membranes were incubated with the following primary antibodies at 4°C overnight: integrin β1 (1:1000, cat.24693, abcam), RhoA (1:1000, cat.6352, Affinity), phosphorylation of RhoA (GTP‐RhoA) (1:500, cat.211164, abcam), JNK (1:1000, cat. AF6319, Affinity), p‐JNK (1:1000, cat. AF3320, Affinity), ICAM‐1 (1:1000, cat. DF7413, Affinity) and VCAM‐1 (1:1000, cat.DF6082, Affinity), NF‐κB P65(1:1000, AF5006, Affinity), PhosphoNFκB P65(1:1000, AF2006, Affinity), IKKα (1:1000, AF6012, Affinity), IKKβ (1:1000, bs‐4880R, Bioss, China), Phospho‐IKKβ(1:1000, bs‐3232R, Bioss, China).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Antioxidants

Article Title: Gastro-Protective Effects of Albizia anthelmintica Leaf Extract on Indomethacin-Induced Gastric Ulcer in Wistar Rats: In Silico and In Vivo Studies

doi: 10.3390/antiox10020176

Figure Lengend Snippet: Effects of indomethacin alone and with oral pre-treatments of famotidine or A. anthelmintica (200, 100 mg/kg) on ( A ) gastric inhibitor of nuclear factor kappa-B kinase subunit beta (IKKB), ( B ) gastric nuclear factor KB (NF-κB), ( C ) gastric TNF-α, and ( D ) gastric IL-6 in rats. Statistical analyses were carried out by one-way ANOVA followed by Tukey’s post hoc test, mean ± SEM. * Significantly different from control group at p < 0.05. @ Significantly different from indomethacin alone group at p < 0.05.

Article Snippet: Primary rabbit polyclonal antibodies against inhibitor of nuclear factor kappa-B kinase subunit beta (IKK-β, catalogue number: PA2036-1) and rabbit monoclonal antibodies against β-actin (catalogue number: M01263) were purchased from Boster Biological Technology (Pleasanton, CA, USA) and diluted at 1:1000 and 1:5000, respectively.

Techniques: Control