Journal: European journal of immunology

Article Title: Sestrin1 inhibits oxidized low-density lipoprotein-induced activation of NLRP3 inflammasome in macrophages in a murine atherosclerosis model.

doi: 10.1002/eji.201948427

Figure Lengend Snippet: Figure 4. The effect of SESN1 on oxLDL-induced NF-κB signaling. (A) qPCR assay on mRNA levels of indicated cytokines normalized to β-actin in macrophages after 24-h treatment with 50 μg/mL oxLDL. LC: LC-infected macrophages. LS1: LS1-infected macrophages. V: vehicle; oxLDL: oxLDL treatment. N = 5 mice pooled from four experiments. One-way ANOVA with Tukey test. (B–D) Immunoblotting images of IKKβ phosphorylation (B), IκBα expression (C), and the expression of nuclear NF-κB p50 and p65 (D) in macrophages after 24-h treatment with oxLDL. β-Actin is the loading control. (E–H) Statistics for IKKβ phosphorylation (E), IκBα expression (F), and the expression of nuclear NF-κB p50 (G) and p65 (H) in macrophages. N = 4–5 mice pooled from three or four experiments. Mann–Whitney test. *p < 0.05; **p < 0.01; ***p < 0.001. Error bars show mean ± SD.

Article Snippet: IκBα antibody (4814), NF-κB p65 antibody (8242), phospho-IKKα/β (Ser176/180) antibody (2694), and IKKβ antibody (8943) were purchased from Cell Signaling Technology.

Techniques: Infection, Western Blot, Phospho-proteomics, Expressing, Control, MANN-WHITNEY

Journal: Journal of Periodontal & Implant Science

Article Title: Interleukin-6 regulates human ODAM gene expression in gingival epithelial cells

doi: 10.5051/jpis.2402980149

Figure Lengend Snippet: Protein levels were assessed using Western blotting following stimulation with IL-6 (10 ng/mL) for durations of 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, and 24 hours in Ca9-22 cells. Densitometric analyses of the Western blot results for ODAM, p-STAT3, STAT3, p-p65, p65, p-IKKα/β, IKKβ, and IKKα are presented. The intensity of the α-tubulin band was normalized to 1, and graphs were generated to illustrate the relative intensities of the other protein bands. IL-6: interleukin-6, ODAM: odontogenic ameloblast-associated protein, STAT3: signal transducer and activator of transcription 3, p-STAT3: phosphorylated STAT3, p65: nuclear factor-kappa B p65, p-p65: phosphorylated p65, IKKα: IκB kinase α, IKKβ: IκB kinase β, p-IKKα/β: phosphorylated IκB kinase α/β. The data represent the mean ± standard deviation from 3 independent experiments, with * P <0.05 and ** P <0.01 indicating statistical significance.

Article Snippet: These membranes were incubated with primary antibodies: anti-ODAM (PA5-100656; Invitrogen), anti-signal transducer and activator of transcription (STAT) 3 (10253-2-AP; Proteintech, Rosemont, IL, USA), anti-phospho-STAT3 (Tyr705) (ZRB1006; Sigma-Aldrich, St. Louis, MO, USA), anti-nuclear factor-kappa B (NF-κB) p65 (AF5006; Affinity Biosciences, Cincinnati, OH, USA), anti-phosphorylated (phospho-)NF-κB p65 (Ser536) (AF2006; Affinity Biosciences), anti-IκB kinase α (IKKα) (ab32041; Abcam, Cambridge, UK), anti-IκB kinase β (IKKβ) (#2684; Cell Signaling Technology, Danvers, MA, USA), anti-phospho-IKKα/β (Ser176/180) 16A6 (#2697; Cell Signaling Technology), and anti-α-tubulin (sc-5286; Santa Cruz Biotechnology).

Techniques: Western Blot, Generated, Standard Deviation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: TANK-binding kinase 1 (TBK1) controls cell survival through PAI-2/serpinB2 and transglutaminase 2.

doi: 10.1073/pnas.1119296109

Figure Lengend Snippet: Fig. 1. TBK1 protects MEFs from TNF-induced apoptosis and modulates IKK-dependent phosphorylation of RelA/p65. (A) Wt and Tbk1−/−MEFs were treated for 24 h with TNF (25 ng/mL) alone or for 3 h with TNF in the presence of CHX (10 μg/mL). Apoptotic cells were detected by TUNEL assays. The bars represent averages ± SD of three different experiments. Approximately 1,200 cells were counted in each experiment. (B) MEFs treated with TNF and CHX for the indicated times were analyzed for caspase-8 and caspase-3 activation by immunodetection of their cleaved forms. (C) Cell extracts prepared from Tbk1−/−

Article Snippet: Antibodies against RelA/p65 (S536) (no. 3031), phospho-IκBα (no. 9241), phospho-MAPKs (no. 9910), phospho-cJun (no. 9261), ERK, p38 (no. 9212), TBK1 (no. 3012), Bcl-XL (no. 2764), c-IAP1 (no. 4952), mBID (no. 2003), caspase-3 (no. 9662), cleaved caspase-3 (no. 9661), and cleaved PARP (no. 9544) were from Cell Signaling; antibodies to IKKγ (no. 557383), JNK1 (no. 551196), XIAP (no. 610716), CD95/Fas receptor (554254), caspase-8 (no. 559932), and procaspase-3 (no. 65906E) were from BD Transduction Laboratories; antibodies to IKKα (no. IMG-136A), IKKβ (no. IMG-129A), TBK1 (IMG-139A), and IκBα (no. IMG-127A) were from Imgenex; antibodies against RelA (sc-372 and sc-372G), RelB, cRel, IκBβ, HSP60, and PAI-2 (sc-25746) were from Santa Cruz Biotechnology; anti-p65/RelA (CT), anti–phospho-ATF-2 (no. 05–891), anti-TG2 (no. 06–471), and anti-caspase-1 (no. 06–503) were from Upstate Biotechnology; anti–CREB-1 (AB3006) was from Millipore; anti-FLIPα (CT; no. 1161) was from ProSci, anti-TG2 (AB-4) was from Neomarkers (Lab Vision), anti-HA (clone 3F10) was from Roche, anti-cAIP2 was from R&D Systems, and anti-CD31 was from Abcam.

Techniques: Phospho-proteomics, TUNEL Assay, Activation Assay, Immunodetection