Journal: PLoS ONE

Article Title: Proinflammatory Cytokines and Bile Acids Upregulate ΔNp73 Protein, an Inhibitor of p53 and p73 Tumor Suppressors

doi: 10.1371/journal.pone.0064306

Figure Lengend Snippet: A. Dynamics of IKKα/β phosphorylation in BA/A-treated cells. SK-GT-4 cells were treated with 100 µM BA cocktail, pH 4.0. Extracts were collected at the indicated time points and analyzed by Western blotting. Early (5–30 minutes) and late (4–24 hours) time points are shown. B. IKK kinase inhibitor Bay11–7085 downregulates the ΔNp73 protein. SK-GT-4 cells were pretreated with Bay11–7085 (10 µM) for 1 hour, treated with BA/A (100 µM) for 30 minutes and then incubated for an additional 7 hours in the presence of the indicated inhibitor. Total cell extracts were analyzed by Western blotting. C. Inhibition of IKK kinases by siRNA leads to downregulation of the ΔNp73 protein in BA/A-treated cells. SK-GT-4 cells were transfected with IKKα- or IKKβ- specific siRNA for 48 hours, treated with BA/A (100 µM) for 30 minutes, and then collected 7 hours after treatment. Levels of ΔNp73 were analyzed by Western blotting. D. Transfection of constitutively active IKKα and IKKβ mutants leads to upregulation of the ΔNp73 protein. SK-GT-4 cells were transfected with the indicated mutants for 24 hours and analyzed by Western blotting.

Article Snippet: Antibodies for the following proteins were used: ΔNp73 (N-16), c-Abl (K-12) and phospho-p73 (Tyr99) from Santa Cruz Biotechnology; p73 from Bethyl Laboratories, phospho-c-Abl (Tyr412), phospho-serine (PSR-45) and FLAG-tag (M2) from Sigma-Aldrich; Phospho-IKKα/β (16A6), IKKα (#2682), IKK β (L570), phospho-Aurora A (Thr288) (C39D8), Aurora A (1G4), DYKDDDDK tag, β-actin (13E5), phospho-p38 MAPK (Thr180/Tyr182), and p38 MAPK (#9212) from Cell Signaling, p73 (Ab2) and phosphotyrosine (4G10) from Millipore.

Techniques: Western Blot, Incubation, Inhibition, Transfection

Journal: Oncology Letters

Article Title: Diosmetin triggers cell apoptosis by activation of the p53/Bcl-2 pathway and inactivation of the Notch3/NF-κB pathway in HepG2 cells

doi: 10.3892/ol.2016.5347

Figure Lengend Snippet: DIOS downregulates Notch3 signaling and inhibits the NF-κB pathway by inactivation of its upstream regulator IKKα/IKKβ. (A) Notch3, cleaved Notch3 and NF-κB were detected by immunoblotting. (B) The p53 cellular location and protein level was analyzed by immunofluorescence (magnification, ×200). (C) IKKα, IKKβ and NF-κB protein level alterations were determined following DIOS stimulation. DIOS, diosmetin; NF, nuclear factor; IKK, IκB kinase.

Article Snippet: Antibodies specific for the following proteins were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA) and used at a 1:1,000 dilution: Notch3/cleaved Notch3 (#5276), NF-κB (#8242), p53 (#2527), PARP (#9532), IκB kinase (IKK)α (#11930), IKKβ (#8943) and GAPDH (#2118).

Techniques: Western Blot, Immunofluorescence