Journal: Oncotarget

Article Title: Selinexor, a Selective Inhibitor of Nuclear Export (SINE) compound, acts through NF-κB deactivation and combines with proteasome inhibitors to synergistically induce tumor cell death

doi: 10.18632/oncotarget.12428

Figure Lengend Snippet: A. U-2 OS cells were stimulated with or without 20ng/mL TNFα for 2 hours before being treated with vehicle, 100nM or 1μM selinexor for the next 24 hours. Western blot of phospho-IκB-α and phosphor-NF-κB p-65 shows that selinexor reverts the pro-inflammatory effects of TNFα and selinexor also increased cellular levels of IκB-α. Selinexor induces XPO1 degradation. It is mediated by the proteasome degradation pathway (TK and YL, not shown). B. IKKβ kinase activity was analyzed by in vitro kinase assay using recombinant IKKβ, recombinant IκB-α substrate containing serine 32/36 residues and selinexor at different concentrations. IKKβ kinase activity was detected using a phosphorylation specific IκB-α antibody. Selinexor had no inhibitory effects on IKKβ kinase activity. The pan-kinase inhibitor K252A was used as a positive control for the assay. C. Immunofluorescence staining of IκB-α after treatment with 20ng/mL TNFα or/and 1μM selinexor for 24 hours. Selinexor induced nuclear localization of IκB-α in the presence or absence of TNFα. D. Cellular fractionation of U-2 OS cells shows similar increased nuclear levels of IκB-α and NF-κB p65 upon selinexor treatment even in the presence of TNFα. Lamin A/C was used as nuclear protein marker; GAPDH as a cytosolic protein marker. E. IκB-α immunoprecipitation (IP) and Western blotting of cytoplasmic and nuclear fractions of U-2OS cells treated with selinexor and TNFα shows that IκB-α binds to NF-κB p65 subunit both in the nucleus and cytoplasm.

Article Snippet: Activity of IKKβ was quantified by IKKβ kinase assay/inhibitor screening kit (MBL #CY-1178) using recombinant IκB-α polypeptide, containing 2 serine residues, which are substrates of IKKβ.

Techniques: Western Blot, Activity Assay, In Vitro, Kinase Assay, Recombinant, Phospho-proteomics, Positive Control, Immunofluorescence, Staining, Cell Fractionation, Marker, Immunoprecipitation

Journal: bioRxiv

Article Title: Mechanism of negative regulation of NF-κB by N4BP1

doi: 10.1101/2020.10.21.349357

Figure Lengend Snippet: a , Immunoblot analysis of phosphorylated (p-) IKKα/β, p65, p38, and c-Jun in lysates of wild-type and N4bp1 -/- peritoneal macrophages stimulated with Pam3CSK4 (40 ng/ml) for the indicated times. Relative densitometric measurements are indicated below. b , Detection of the endogenous NEMO-IKKβ association in wild-type and N4bp1 -/- peritoneal macrophages stimulated with Pam3CSK4 for the indicated times, assessed by immunoprecipitation (IP) with rabbit IgG as a control, or with anti-NEMO, followed by immunoblot analysis with anti-IKKβ or anti-NEMO. c , In vitro IKKα/β kinase assay. The IKK complex was immunoprecipitated from N4bp1 +/+ or N4bp1 -/- peritoneal macrophages with anti-NEMO. Cells were treated with or without Pam3CSK4 (40 ng/ml) for 30 min before collection. d , ChIP assay and qPCR of Il6 promoter DNA in peritoneal macrophages 2 h after Pam3CSK4 stimulation. Cell lysates were immunoprecipitated with rabbit IgG, anti-p65, or anti-acetyl-histone H4. e , RT-qPCR analysis of Il1β, Il6, Il10, Il12p40, Tnf , and Ccl5 in N4bp1 +/+ and N4bp1 -/- peritoneal macrophages stimulated with R848. f , Left , Luciferase reporter activity dependent on the indicated promoters (X-axis) in wild-type or N4BP1 -/- HEK293T cells. Right , Immunoblot analysis of N4BP1 expression in wild-type and N4BP1 -/- HEK293T cells. g , Immunoblot analysis of IκBα in lysates of wild-type and N4BP1 -/- HEK293T cells treated with cycloheximide (CHX, 20 μg/ml) for the indicated times. Relative densitometric measurements of IκBα are indicated below. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (two-tailed Student’s t -test). Data are representative of two independent experiments (mean ± s.d. in d , e , f ).

Article Snippet: The IKKα/β kinase activity in the IP complex was tested by using IKKβ Kinase Enzyme Kit (Promega) and following the manufacturer’s instructions.

Techniques: Western Blot, Immunoprecipitation, In Vitro, Kinase Assay, Quantitative RT-PCR, Luciferase, Activity Assay, Expressing, Two Tailed Test