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: Molecular Therapy Oncology

Article Title: Oncolytic virus V937 in combination with PD-1 blockade therapy to target immunologically quiescent liver and colorectal cancer

doi: 10.1016/j.omton.2024.200807

Figure Lengend Snippet: List of primer assay used for RNA expression analysis

Article Snippet: IKBKB , Hs00233287_m1 , TLR9 , Hs00370913_s1.

Techniques: RNA Expression

Journal: International Journal of Biological Sciences

Article Title: Defective neurite elongation and branching in Nibp/Trappc9 deficient zebrafish and mice

doi: 10.7150/ijbs.78489

Figure Lengend Snippet: Key Resource Table

Article Snippet: Rabbit anti-TRAPPC9 , Proteintech , Cat #:16014-1-AP; RRID: AB_2256482.

Techniques: Sequencing, Reverse Transcription, SYBR Green Assay, Recombinant, Software

Journal: Cell reports

Article Title: Persistent NF-κB activation in muscle stem cells induces proliferation-independent telomere shortening

doi: 10.1016/j.celrep.2021.109098

Figure Lengend Snippet:

Article Snippet: Primers purchased from Applied Biosystems and included IKBKB-FAM (Gene assay ID: Mm01222247_m1), CHUK-FAM (Mm00432529_m1), IKBKG-FAM (Mm00494927_m1), XRCC5-FAM (Mm00550142_m1), XRCC6-FAM (Mm00487458_m1), TERF2-FAM (Mm01253555_m1), TERF1-FAM (Mm00436928_m1), TERF2IP-FAM (Mm01243676_m1), TPP1-FAM (Mm00487016_m1), TINF2-FAM (Mm00461166_g1), Pot1B-FAM (Mm01278790_m1), Tert-FAM (Mm00436931_m1), and GAPDH-VIC (4352339e).

Techniques: Recombinant, Electron Microscopy, Plasmid Preparation, Avidin-Biotin Assay, Blocking Assay, Saline, Lysis, Imaging, Kinase Assay, Staining, Generated, Software, Microscopy

Journal: Oncotarget

Article Title: Down-regulation of IKKβ expression in glioma-infiltrating microglia/macrophages is associated with defective inflammatory/immune gene responses in glioblastoma

doi:

Figure Lengend Snippet: A. Total RNA and proteins were isolated from the same tumor samples after TRIzol procedure. The level of IKBKB mRNA in juvenile PA and GBM was determined by qPCR with specific TaqMan probes. Lower ΔCT is consistent with higher gene expression. B. The IKKβ level in protein extracts obtained from JPA and in GBM (16 samples/group). After stripping the immunoblots were re-probed with anti-β-actin antibody to ensure equal protein loading. C. Densitometric analysis of the IKKβ immunoblots (21 samples/group) has been performed and the results are shown as IKKβ/β-actin ratio in a given sample.

Article Snippet: Reaction volume (10 μl) consisted of cDNA equivalent to 18.75 ng RNA, TaqMan Fast Universal PCR Master Mix 2x (Applied Biosystems, Darmstadt, Germany) and IKBKB probe (Hs00826074_m1, Applied Biosystems, Darmstadt, Germany), the primer set for CXCL14 expression (Qiagen NM_004887) or specific primers ( ).

Techniques: Isolation, Gene Expression, Stripping Membranes, Western Blot

Journal: Oncotarget

Article Title: Down-regulation of IKKβ expression in glioma-infiltrating microglia/macrophages is associated with defective inflammatory/immune gene responses in glioblastoma

doi:

Figure Lengend Snippet: A, B. Images of double staining for CD11b + (red) and IKKβ (brown) performed on sections of GBM (A) and JPA (B); scale bars 100 μm (insets: 50 μm). C. Microglia/macrophages were separated from freshly resected tumors using a magnetic-bead-conjugated anti-CD11b antibody. Purity of the positive fraction stained with anti-CD11b-PE antibody after magnetic separation was 93%. D–E. Quantification of the IKBKB (D) and M1/M2 marker gene expression (E) in CD11b + cells isolated from low and high grade gliomas. Lower ΔCT is consistent with higher gene expression. Statistical analysis was done with the Wilcoxon testand t test, respectively.

Article Snippet: Reaction volume (10 μl) consisted of cDNA equivalent to 18.75 ng RNA, TaqMan Fast Universal PCR Master Mix 2x (Applied Biosystems, Darmstadt, Germany) and IKBKB probe (Hs00826074_m1, Applied Biosystems, Darmstadt, Germany), the primer set for CXCL14 expression (Qiagen NM_004887) or specific primers ( ).

Techniques: Double Staining, Staining, Marker, Gene Expression, Isolation

Journal: Oncotarget

Article Title: Down-regulation of IKKβ expression in glioma-infiltrating microglia/macrophages is associated with defective inflammatory/immune gene responses in glioblastoma

doi:

Figure Lengend Snippet: A. Double immunofluorescence staining of the IKKβ and Iba1 followed by staining with DAPI was performed on sections from sham-operated rats (upper), rats 15 days after C6 glioma cell implantation (middle) and rats 24 h after 90 min ischemia/reperfusion (lower) ( n = 3/group). The increased IKKβ immunoreactivity is visible in Iba1 positive cells present in the cerebral cortex of rats subjected to transient MCAo. B. The Ikbkb expression was strongly induced in rat primary microglial cultures 6 h after stimulation with 100 ng/ml lipopolysaccharide (LPS) but not after exposure to glioma conditioned medium (GCM). Representative immunoblot shows the increased level of phospho-IκB 6 h after treatment of microglia with LPS but not in GCM-treated cultures. The increased level of phospho-IκB is indicative of IKKβ signaling activation.

Article Snippet: Reaction volume (10 μl) consisted of cDNA equivalent to 18.75 ng RNA, TaqMan Fast Universal PCR Master Mix 2x (Applied Biosystems, Darmstadt, Germany) and IKBKB probe (Hs00826074_m1, Applied Biosystems, Darmstadt, Germany), the primer set for CXCL14 expression (Qiagen NM_004887) or specific primers ( ).

Techniques: Double Immunofluorescence Staining, Staining, Expressing, Western Blot, Activation Assay