Journal: Molecular Cancer

Article Title: The inflammatory kinase IKKα phosphorylates and stabilizes c-Myc and enhances its activity

doi: 10.1186/s12943-021-01308-8

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Article Snippet: IKKα (D3W6N) Rabbit mAb (1:200) , Cell Signaling Technology, Frankfurt, Germany , Cat#: 61294; RRID: AB_2799606.

Techniques: Staining, Luciferase, Cloning, Mutagenesis, Recombinant, Plasmid Preparation, Software

Journal: The Journal of Clinical Investigation

Article Title: Selective inhibition of long isoforms of phosphodiesterase 4D mitigates liver fibrosis in mouse models

doi: 10.1172/JCI182571

Figure Lengend Snippet: ( A ) A volcano plot depicting the differentially expressed genes in LX-2 cells cotreated with TGFb and D159687 versus TGFb-treated LX-2 cells. ( B ) Gene-ontology analysis of molecular functions. ( C ) Visualization of RNA-seq coverage of CCL2 . ( D and E ) LX-2 cells were treated with 2 ng/ml TGFb for 24 hours in the presence of 10 μM D159687. CCL2 and CCL7 mRNA levels were determined via RT-qPCR. ( F ) Phosphorylated ERK was visualized by immunoblotting. ( G ) The AP-1 transcriptional activity was measured using reporter assay in AP-1-stable reporter LX-2 cells. ( H ) Effect of PDE4D on the chemotaxis of THP-1 cells in response to TGFb-mediated LX-2 CM. The migratory cells were quantified according to the fluorescence intensity. ( I ) Kupffer cells were treated with 1 μg/ml LPS in the presence of 10 μM D159687 for 24 hours. Ccl2 mRNA levels were determined by RT-qPCR. ( J and K ) Phosphorylated IKK and NF-κB were visualized using antibodies specific for Phospho-IKKa/b (Ser176/180) and Phospho-NF-κB p65 (Ser536). ( L ) The NF-κB transcriptional activity in stable reporter Kupffer cells with the NF-κB response element was evaluated via a reporter assay. ( M ) Effect of PDE4D on the chemotaxis of THP-1 cells in response to LPS-mediated Kupffer cell CM. ( N ) Inflammatory cytokines, including Il-1β and Tnfa , were determined in MoMs cocultured with Kupffer cells. All values are presented as the mean ± SEM of at least 3 independent experiments. 1-way ANOVA with Tukey’s post hoc test for multiple comparisons was used for statistical analysis in D , E , G – I , and L – N . * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Primary antibodies were used at 1:1,000 dilution, including COL1A1 (#84336, Cell signaling); ACTA2 (#A2547, Sigma-Aldrich); COL1A1 (#84336, Cell signaling); p-SMAD2 (#3108, Cell signaling); p-SMAD3 (#ab52903, abcam); SMAD2/3 (#8685, Cell signaling); SMAD4 (#46535, Cell signaling); LAMIN (#13435, Cell signaling); p-ERK (#4370, Cell signaling); ERK (#4696, Cell signaling); p-IKK (#2697, Cell signaling); IKK (#11930, Cell signaling); p-NF-κB p65 S536 (#3033, Cell signaling); NF-κB P65 (#8242, Cell signaling); p-AKT (#4060, Cell signaling); AKT (#4691, Cell signaling); p-FOXO1 S256 (#9461, Cell signaling); p-FOXO3a S253 (#13129, Cell signaling); FOXO1 (#2880, Cell signaling); p18 INK4C (#2896, Cell signaling); p21 Waf1/Cip1 (#2947, Cell signaling); CDK4 (#12790, Cell signaling); CDK6 (#3136, Cell signaling); PARP (#9542, Cell signaling); CASPASE3 (#14220, Cell signaling); Integrin a5 (#ab150361, abcam); p-FAK Y397 (#8556, Cell signaling); p-FAK Y925 (#3284, Cell signaling); FAK (#13009, Cell signaling); FLAG (#F1804, Sigma-Aldrich); a-tubulin (sc-8035, Santa Cruz).

Techniques: RNA Sequencing, Quantitative RT-PCR, Western Blot, Activity Assay, Reporter Assay, Chemotaxis Assay, Fluorescence

Journal: PLoS ONE

Article Title: Kaposi’s Sarcoma Associated Herpesvirus Encoded Viral FLICE Inhibitory Protein K13 Activates NF-κB Pathway Independent of TRAF6, TAK1 and LUBAC

doi: 10.1371/journal.pone.0036601

Figure Lengend Snippet: A. Co-immunoprecipitation assay showing NEMO is essential for mediating the interaction of K13 with IKK1 and IKK2. FLAG-tagged K13 was immunoprecipitated from WT and NEMO-deficient Jurkat cells using control (C) and FLAG (F) antibody beads and presence of co-immunoprecipitated IKK1, IKK2 and NEMO detected by immunoblotting. B. MEFs expressing K13-ER TAM were treated with 4OHT and phosphorylation status of IKK1, IKK2 and IκBα was determined using Pathscan phospho-IKK1 (Ser176/180), phospho-IKK2 (Ser177/181), phosphor-IκBα (Ser32) ELISA kits, respectively.

Article Snippet: The PathScan Phospho-IKKα (Ser176/180), Phospho-IKKβ (Ser177/181) sandwich ELISA Kits and PathScan Phospho-IκBα (Ser32) sandwich ELISA antibody pair (Cell Signaling, Danvers, MA) were used to detect endogenous levels of IKKα, IKKβ and IκBα proteins when phosphorylated at Ser176/180, Ser177/181 and Ser32, respectively.

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Control, Western Blot, Expressing, Phospho-proteomics, Enzyme-linked Immunosorbent Assay