Journal: Cancers

Article Title: YB-1 Mediates TNF-Induced Pro-Survival Signaling by Regulating NF-κB Activation

doi: 10.3390/cancers12082188

Figure Lengend Snippet: Y-box binding protein-1 (YB-1) is essential for tumor necrosis factor (TNF)-induced NF-κB nuclear translocation in bone marrow-derived macrophages. ( A ) Imaging flow cytometry showing one representative cell. The nucleus stained positive for 7-Amino-actinomycin D (7AAD) (red). Staining for NF-κB phospho-p65 activation (pp65) is shown with and without stimulation (green). Colocalization (yellow) indicates nuclear translocation. ( B ) Normalized frequency of wild type (WT) (black) and YB-1 knockout (KO) (Ybx1 ΔLysM ) cells (red) showing nuclear translocation of activated NF-κB p65 with and without TNF stimulation (20 ng/mL).

Article Snippet: The following antibodies were used: anti-NF-κB p65 (6956, Cell Signaling, Frankfurt am Main, Germany), anti-phospho-NF-κB p65 (pp65; 3033, Cell Signaling), anti-YB-1 (Eurogentec, Liège, Belgium), anti-TRAF2 (558,890, BD Biosciences), anti-RIP (610,459, BD Biosciences, Heidelberg, Germany), IκBα (9242, Cell Signaling), anti-p-IκBα (9246, Cell Signaling), anti-TNFR1 (8436, Santa Cruz, CA, USA), anti-vinculin (59,803, Santa Cruz), anti-mono- and polyubiquitinated protein (clone FK2, Enzo), anti-cleaved caspase-3 (9661, Cell Signaling), anti-caspase-8 (4790, Cell Signaling), anti-cleaved caspase-8 (9496, Cell Signaling), and anti-pIKKα/β (2697, Cell Signaling).

Techniques: Binding Assay, Translocation Assay, Derivative Assay, Imaging, Flow Cytometry, Staining, Activation Assay, Knock-Out

Journal: Cancers

Article Title: YB-1 Mediates TNF-Induced Pro-Survival Signaling by Regulating NF-κB Activation

doi: 10.3390/cancers12082188

Figure Lengend Snippet: YB-1 was required for TNF-induced NF-κB activation. ( A ) Bone marrow-derived macrophages (BMDM, WT, and YB-1 KO) were stimulated with TNF (20 ng/mL) for the indication of time periods to activate the NF-κB signaling pathway. Protein expression was analyzed by immunoblotting using the indicated antibodies. Vinculin was used as the loading control. The relative band intensities are indicated. ( B ) Relative YB-1 expression is shown for three independent experiments. WT (white) and KO (black bars). ( C ) Control and YB-1 knockdown (KD) cells (THP-1 and U937) were stimulated with TNF (20 ng/mL) to activate tumor necrosis factor receptor 1 (TNFR1). Proteins were analyzed by immunoblotting for expression of the indicated proteins. Vinculin was used as loading control. Control (CTRL; white) and knockdown (KD; black bars). ( D ) Relative YB-1 expression in KD cells is shown for three independent experiments. ( E ) Relative pp65, phospho- inhibitor of NF-κB alpha (p-IκBα), phospho-IkB kinase α/β (pIKKα/β), and TNF receptor-associated factor 2 (TRAF2) expression in WT and KD THP-1 and U937 cells are shown for three independent experiments. Control (CTRL; white) and knockdown (KD; black bars). Error bars specify the standard error of the mean (SEM). Statistical significance was calculated using an unpaired t -test, n = 3. Data represent the mean ± SEM. * p < 0.05, ** p < 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: The following antibodies were used: anti-NF-κB p65 (6956, Cell Signaling, Frankfurt am Main, Germany), anti-phospho-NF-κB p65 (pp65; 3033, Cell Signaling), anti-YB-1 (Eurogentec, Liège, Belgium), anti-TRAF2 (558,890, BD Biosciences), anti-RIP (610,459, BD Biosciences, Heidelberg, Germany), IκBα (9242, Cell Signaling), anti-p-IκBα (9246, Cell Signaling), anti-TNFR1 (8436, Santa Cruz, CA, USA), anti-vinculin (59,803, Santa Cruz), anti-mono- and polyubiquitinated protein (clone FK2, Enzo), anti-cleaved caspase-3 (9661, Cell Signaling), anti-caspase-8 (4790, Cell Signaling), anti-cleaved caspase-8 (9496, Cell Signaling), and anti-pIKKα/β (2697, Cell Signaling).

Techniques: Activation Assay, Derivative Assay, Expressing, Western Blot, Control, Knockdown

Journal: International Journal of Molecular Sciences

Article Title: Sitagliptin Induces Tolerogenic Human Dendritic Cells

doi: 10.3390/ijms242316829

Figure Lengend Snippet: The effect of sitagliptin (500 µg/mL) on the activation of p65 NF-kB and p38 MAPK in MoDCs. The activation of p65 NF-kB and p38 MAPK was analyzed using Western blot by the phosphorylated ( p ) and total forms of these molecules, as described in the Materials and Methods. Values are from two different experiments ( A ); supported by the original blots ( B ). * p < 0.05; ** p < 0.01 compared with corresponding controls as indicated by bars. GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) was used as a loading control.

Article Snippet: Western blotting was performed overnight at 4 °C with antibodies against: p38 MAPK (D13E1) (1:1000, Cell Signaling, Danver, MA, USA, #8690), phospho-p38 MAPK (Thr180/Tyr182) (1:1000, Cell Signaling, Danver, MA, USA, #9211), NFκB p65 (C-20): sc-372 (1:1000, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and Phospho-NF-κB p65 (pp65) (Ser536) (93H1) (1:1000, Cell Signaling, Danver, MA, USA, Rabbit mAb #3033).

Techniques: Activation Assay, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Sitagliptin Induces Tolerogenic Human Dendritic Cells

doi: 10.3390/ijms242316829

Figure Lengend Snippet: The effect of sitagliptin (500 µg/mL) on the activation of p65 NF-kB and p38 MAPK in MoDCs. The activation of p65 NF-kB and p38 MAPK was analyzed using Western blot by the phosphorylated ( p ) and total forms of these molecules, as described in the Materials and Methods. Values are from two different experiments ( A ); supported by the original blots ( B ). * p < 0.05; ** p < 0.01 compared with corresponding controls as indicated by bars. GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) was used as a loading control.

Article Snippet: Western blotting was performed overnight at 4 °C with antibodies against: p38 MAPK (D13E1) (1:1000, Cell Signaling, Danver, MA, USA, #8690), phospho-p38 MAPK (Thr180/Tyr182) (1:1000, Cell Signaling, Danver, MA, USA, #9211), NFκB p65 (C-20): sc-372 (1:1000, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and Phospho-NF-κB p65 (pp65) (Ser536) (93H1) (1:1000, Cell Signaling, Danver, MA, USA, Rabbit mAb #3033).

Techniques: Activation Assay, Western Blot, Control