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: PLoS ONE

Article Title: The Role of IKKβ in Venezuelan Equine Encephalitis Virus Infection

doi: 10.1371/journal.pone.0086745

Figure Lengend Snippet: U87MG cells were infected with TC-83 or UV-TC-83 (MOI: 5) or the cells were treated with Poly I∶C (10 µg/mL) or Imiquimod (2 µg/mL). Cells were lysed 24 hours post-infection and post treatment and protein extracts were quantified. Two milligrams of total protein was subjected to chromatography using a Superose 6 HR 10/30 size-exclusion chromatography column and an AKTA purifier system. Every 5th fraction was acetone precipitated using 4 volumes of ice-cold 100% acetone and incubated for 15 minutes on ice. Lysates were centrifuged at 4°C for 10 minutes at 12,000 rpm, supernatants were removed, and the pellets were allowed to dry for a few minutes at room temperature. The pellets were resuspended in Laemmli buffer and analyzed by immunoblotting using IKKα (A), IKKβ (B), IKKγ (C) and β-actin (D) antibodies. Every fifth fraction ranging from 2.2 mDa to 100 kDa is represented on the western blot. Smaller IKKβ complexes (highlighted in the red box) in the higher fractions suggests a rearrangement of the IKKβ complex in TC-83 infected cells.

Article Snippet: Primary antibodies to VEEV Capsid (BEI Resources, NR 9403), VEEV Glycoprotein (BEI Resources, NR 9404), total p65 (Santa Cruz Biotechnology, Catalogue No. sc-7151), phospho-p65 (ser536) (Santa Cruz Biotechnology, Catalogue No. sc-33020), phospho-IκBα (Santa Cruz Biotechnology, Catalogue No. sc-21869), total IκBα (Santa Cruz Biotechnology, Catalogue No. sc-847), IKKα (Santa Cruz Biotechnology, Catalogue No. sc-7218), IKKβ (Santa Cruz Biotechnology, Catalogue No. sc-7329), IKKγ (Santa Cruz Biotechnology, Catalogue No. sc-8330), HA probe (F-7) (Santa Cruz Biotechnology, Catalogue No. sc-7392), V5 (AbD Serotec, Catalogue No. MCA 1360) and HRP conjugated actin (Abcam, Catalogue No. ab49900) were used according to manufacturer's instructions.

Techniques: Infection, Chromatography, Size-exclusion Chromatography, Incubation, Western Blot

Journal: Cellular signalling

Article Title: Minocycline Inhibits PDGF-BB-induced Human Aortic Smooth Muscle Cell Proliferation and Migration by reversing miR-221- and -222-mediated RECK suppression

doi: 10.1016/j.cellsig.2019.01.014

Figure Lengend Snippet: A, PDGF-BB induces time-dependent NF-κB activation. Quiescent SMC were incubated with PDGF-BB (10 ng/ml). At the indicated time periods, activation of NF-κB was analyzed by immunoblotting using antibodies that specifically detect phosphorylated p65 at Ser536. B, PDGF-BB induces time-dependent AP-1 activation. Quiescent SMC incubated as in A were analyzed for AP-1 activation by immunoblotting using antibodies that specifically detect phosphorylated c-Jun at Ser73. C, Silencing IKKβ or pre-treatment with minocycline inhibit PDGF-BB-induced NF-κB activation. SMC incubated with lentiviral IKKβ shRNA (moi0.5 for 48 h) were made quiescent and treated with PDGF-BB (10 ng/ml for 30 min). In a subset of experiments, quiescent SMC were incubated with minocycline (10 μM for 15 min) and then treated with PDGF-BB (10 ng/ml for 30 min). Activation of NF-κB was analyzed as in A. D, Silencing JNK2 or pre-treatment with minocycline inhibits PDGF-BB-induced AP-1 activation. SMC incubated with lentiviral JNK2 shRNA (moi 0.5 for 48 h) were made quiescent and treated with PDGF-BB (10 ng/ml for 30 min). In a subset of experiments, quiescent SMC were incubated with minocycline (10 mM for 15 min) and then treated with PDGF-BB (10 ng/ml for 30 min). Activation of AP-1 was analyzed as in B. Silencing IKKβ and JNK2 was confirmed by immunoblotting. JNK2 and IKKβ served as off-targets in IKKβ and JNK2 silenced cells, respectively (right hand panels in C and D). Tubulin served as a loading control. Bar graphs at the bottom of panels in A-D represent densitometric analyses from three independent experiments. *P<0.05 control, †P<0.05 versus PDGF-BB (n=3).

Article Snippet: Adeno- and Lentiviral transduction The following lentiviral shRNA vectors against IKKβ (#sc-35645-V), p65 (#sc-29410-V), JNK2 (#sc-39101-V), and c-Jun (#sc-29223-V) were purchased from Santa Cruz Biotechnology, Inc. Lentiviral shRNA against RECK (SHCLNV- {"type":"entrez-nucleotide","attrs":{"text":"NM_021111","term_id":"1519313840","term_text":"NM_021111"}} NM_021111 ; TRCN0000376461) and eGFP (SHC005V) were purchased from Sigma-Aldrich.

Techniques: Activation Assay, Incubation, Western Blot, shRNA

Journal: Cellular signalling

Article Title: Minocycline Inhibits PDGF-BB-induced Human Aortic Smooth Muscle Cell Proliferation and Migration by reversing miR-221- and -222-mediated RECK suppression

doi: 10.1016/j.cellsig.2019.01.014

Figure Lengend Snippet: A, B, PDGF-BB induces miR-221 (A) and miR-222 (B) expression via IKKβ, NF-κB, JNK and AP-1. Quiescent SMC incubated with PDGF-BB (10 ng/ml) for lh were analyzed for miR-221 (A) and miR-222 (B) expression by TaqMan® Advanced miRNA assays. The results were normalized to corresponding U6 expression. In a subset of experiments, SMC were incubated with lentiviral IKKβ, p65, JNK2 or c-Jun shRNA (moi0.5 for 48 h), made quiescent and then treated with PDGF-BB addition. C, D, miR-221 and miR-222 mediate PDGF-induced SMC migration (C) and proliferation (D), without affecting cell viability (E). SMC were transduced with miR-221 or miR-222 inhibitors prior to the addition of PDGF-BB (10 ng/ml). Cell migration was analyzed after 18 h using transwell migration assays (C). Cell proliferation was analyzed after 48h by CyQUANT® Cell Proliferation Assay (D). Cleaved caspase-3 levels, indicative of cells undergoing apoptosis, was analyzed after 8 h by immunoblotting using antibodies that detect both total and cleaved caspase-3 levels (E). Hydrogen peroxide (H2O2, 100 μM) served as a positive control. *P

Article Snippet: Adeno- and Lentiviral transduction The following lentiviral shRNA vectors against IKKβ (#sc-35645-V), p65 (#sc-29410-V), JNK2 (#sc-39101-V), and c-Jun (#sc-29223-V) were purchased from Santa Cruz Biotechnology, Inc. Lentiviral shRNA against RECK (SHCLNV- {"type":"entrez-nucleotide","attrs":{"text":"NM_021111","term_id":"1519313840","term_text":"NM_021111"}} NM_021111 ; TRCN0000376461) and eGFP (SHC005V) were purchased from Sigma-Aldrich.

Techniques: Expressing, Incubation, shRNA, Migration, Transduction, CyQUANT Assay, Proliferation Assay, Western Blot, Positive Control