Journal: Journal of Biological Chemistry

Article Title: Taurocholate Induces Cyclooxygenase-2 Expression via the Sphingosine 1-phosphate Receptor 2 in a Human Cholangiocarcinoma Cell Line

doi: 10.1074/jbc.m115.668277

Figure Lengend Snippet: FIGURE 5. The effect of TCA on activation of IKK/-NF-B pathways in HuCCT-1 cells. A–C, time course of TCA-induced activation of IKK/-NF-B pathways. Cells were cultured in serum-free medium overnight and then treated with TCA (100 M) for different treatment periods (0–24 h). The protein levels of phosphorylated IKK/ (p-IKK/), total IKK/, phosphorylated NF-B p65 (p-NF-B p65), and total NF-B p65 were determined by Western blot analysis. D–F, TCA-induced dose-dependent activation of IKK/-NF-B pathways. Cells were cultured in serum-free medium overnight and then treated with different concentrations of TCA (0, 25, 50, 100, or 200 M) for 4 h. At the end of each treatment, cells were harvested, and total protein was isolated. The protein levels of p-IKK/, total IKK/, p-NF-B p65, and total NF-B p65 were determined by Western blot analysis. A and D, representative images of p-IKK/, total IKK/, p-NF-B p65, and total NF-B p65 are shown. B, C, E, and F, relative protein levels of p-IKK//IKK/ and p-NF-B p65/NF-B p65 were determined. Values represent the mean S.E. of three independent experiments. Statistical significance relative to vehicle control: *, p 0.05; **, p 0.01; ***, p 0.001.

Article Snippet: Primary antibodies against COX-2, p-ERK1/2, ERK1, ERK2, p-Akt, Akt, p-IKK / , IKK / , p-NF- B p65, NF- B p65, p-EGFR, EGFR, -actin, and lamin B, as well as HRP-conjugated secondary antibodies for Western blot were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Cell Culture, Western Blot, Isolation, Control

Journal: Journal of Biological Chemistry

Article Title: Taurocholate Induces Cyclooxygenase-2 Expression via the Sphingosine 1-phosphate Receptor 2 in a Human Cholangiocarcinoma Cell Line

doi: 10.1074/jbc.m115.668277

Figure Lengend Snippet: FIGURE 6. The effect of TCA on nuclear translocation of NF-B. HuCCT1 cells were cultured in serum-free medium overnight and then treated with TCA (100 M)fordifferenttreatmentperiods(0,2,4,8,or24h).Attheendofeachtreatment,cytosolandnuclearproteinswereisolatedasdescribedunder“Experimental Procedures.” Protein levels of NF-B p65 were determined by Western blot analysis. Lamin B and actin were used as loading controls for nuclear and cytosol protein, respectively. A, representative images of the immunoblots for NF-B p65, lamin B, and actin are shown. B, relative protein levels of NF-B p65 in the nucleus and cytosol. Values represent the mean S.E. of three independent experiments. Statistical significance relative to the vehicle control: ***, p 0.001. C and D, immunofluorescence staining of NF-B p65. HuCCT1 cells were cultured in serum-free medium overnight and treated with either 100 M TCA for different treatment periods (0, 2, 4, or 8 h) or different concentrations of TCA (0, 25, 50, or 100 M) for 4 h. At the end of treatment, immunofluorescence staining was performed to detect subcellular location of NF-B p65. Representative images for each group are shown.

Article Snippet: Primary antibodies against COX-2, p-ERK1/2, ERK1, ERK2, p-Akt, Akt, p-IKK / , IKK / , p-NF- B p65, NF- B p65, p-EGFR, EGFR, -actin, and lamin B, as well as HRP-conjugated secondary antibodies for Western blot were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Translocation Assay, Cell Culture, Western Blot, Control, Immunofluorescence, Staining

Journal: Journal of Biological Chemistry

Article Title: Taurocholate Induces Cyclooxygenase-2 Expression via the Sphingosine 1-phosphate Receptor 2 in a Human Cholangiocarcinoma Cell Line

doi: 10.1074/jbc.m115.668277

Figure Lengend Snippet: FIGURE8.TheeffectofJTE-013onS1P-andTCA-inducednucleartranslocationofNF-B.HuCCT1cellswereculturedinserum-freemediumovernightand then treated with either 100 M TCA for different treatment periods (0, 2, 4, 8, or 24 h) or different concentrations of TCA (0, 25, 50, or 100 M) for 4 h. At the end of treatment, immunofluorescence staining was performed to detect subcellular location of NF-B p65. Representative images for each group are shown.

Article Snippet: Primary antibodies against COX-2, p-ERK1/2, ERK1, ERK2, p-Akt, Akt, p-IKK / , IKK / , p-NF- B p65, NF- B p65, p-EGFR, EGFR, -actin, and lamin B, as well as HRP-conjugated secondary antibodies for Western blot were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Immunofluorescence, Staining

Journal: Journal of Biological Chemistry

Article Title: Taurocholate Induces Cyclooxygenase-2 Expression via the Sphingosine 1-phosphate Receptor 2 in a Human Cholangiocarcinoma Cell Line

doi: 10.1074/jbc.m115.668277

Figure Lengend Snippet: FIGURE 7. The effect of chemical antagonist of S1PR2 on S1P- and TCA-induced activation of IKK/-NF-B pathway. HuCCT1 cells were cultured in serum-free medium overnight and pre-treated with JTE-013 (10 M) for 1 h, and then treated with S1P (100 nM) or TCA (100 M) for 4 h. A and B, total protein was isolated, and protein levels of p-IKK/, total IKK/, p-NF-B p65, and total NF-B p65 were determined by Western blot analysis. The relative densities of p-IKK//total IKK/ and p-NF-B p65/total NF-Bp65 were determined. C and D, cytosol and nuclear proteins were isolated as described under “Experi- mental Procedures.” Protein levels of NF-B p65, lamin B, and actin were determined by Western blot analysis. Lamin B and actin were used as a loading control for nuclear and cytosol protein, respectively. A and C, representative images of the immunoblots for p-IKK/, total IKK/, p-NF-B p65, and total NF-B p65 are shown. B, relative protein levels of p-IKK//total IKK/ and p-NF-B p65/total NF-B p65. D, relative protein levels of nuclear NF-B p65/lamin B and cytosol NF-B p65/actin. Values represent the mean S.E. of three independent experiments. Statistical significance relative to the vehicle control: *, p 0.05; **, p 0.01; ***, p 0.001; relative to S1P or TCA treatment group: #, p 0.05; ##, p 0.01; ###, p 0.001.

Article Snippet: Primary antibodies against COX-2, p-ERK1/2, ERK1, ERK2, p-Akt, Akt, p-IKK / , IKK / , p-NF- B p65, NF- B p65, p-EGFR, EGFR, -actin, and lamin B, as well as HRP-conjugated secondary antibodies for Western blot were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Cell Culture, Isolation, Western Blot, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Maackiain dampens osteoclastogenesis via attenuating RANKL‐stimulated NF‐κB signalling pathway and NFATc1 activity

doi: 10.1111/jcmm.15647

Figure Lengend Snippet: Maackiain suppressed the activation of NF‐κB signalling pathway. BMMs were stimulated with RANKL for various periods with or without 40 μmol/L Maackiain. The total protein was examined by Western blot. It indicated that Maackiain could inhibit the degradation of IκB‐α on the time‐points of 10 and 20 min, and dampened the phosphorylation of p65 since the time‐point of 20 min. n = 3; ** P < .01, *** P < .001

Article Snippet: Primary antibody against p‐p65 (Cat#3031S) was obtained from Cell Signalling Technology.

Techniques: Activation Assay, Western Blot, Phospho-proteomics

Journal: bioRxiv

Article Title: Lethal giant larvae gene family ( Llgl1 and Llgl2 ) functions as a tumor suppressor in mouse skin epidermis

doi: 10.1101/2023.03.06.531408

Figure Lengend Snippet: A . RNAs extracted from Llgl1/2 fl/fl and Llgl1/2 -/- primary keratinocytes was analyzed by RNA-Seq (3 samples for each cell type). Significant differences in gene expression (q<0.05) were investigated by Gene Set Enrichment Analysis (GSEA). Two most significantly enriched Hallmark gene sets in Llgl1/2 -/- keratinocytes are Interferons gamma and alpha response sets (q=0.01 and q=0.016). B . Western blot analysis of proteins extracted from primary keratinocytes with indicated genotypes. Note increase in phosphorylation of IKKbeta and RelA in Llgl1/2 -/- and Llgl1/2 -/- /Trp53 +/- , but not in Trp53 +/- primary keratinocytes.

Article Snippet: For Western blotting we used the following antibodies: rabbit anti-LLGL1 (1:20,000, Sigma, HPA023569); rabbit anti-LLGL2 (1:1000, Sigma, HPA022913); rabbit anti-RelA-p65 (1:1000, Cell Signaling, #3033); rabbit anti-p-RelA-p65 (1:1000, Cell Signaling, #8242); mouse anti-aPKC (1:1000, Santa Cruz, sc-17781); rabbit anti-p-IKKα/β (1:500, Cell Signaling, #2697), rabbit anti-IKKβ (1:1000, Cell Signaling, #8943), rabbit anti-p-YAP (1:500, Cell Signaling, #4911), rabbit anti-YAP (1:500, Cell Signaling, #4912), rabbit anti-LATS1/2 (1:1000, AbCam, #ab70565), rabbit anti-p-LATS1/2 (1:500, Cell Signaling, #9159), mouse anti-p-AKT (1:1000, Cell Signaling, #4051), rabbit anti-AKT (1:1000, Cell Signaling, #9272), mouse anti-P-MAPK (1:2000, Sigma #M-8159), rabbit anti-MAPK (1:500, Sigma #M-5670), rabbit anti-TAZ, (1:1000, Sigma, #HPA007415), mouse anti-beta actin (1:5000, Sigma, #A5441).

Techniques: RNA Sequencing, Gene Expression, Western Blot, Phospho-proteomics

Journal: bioRxiv

Article Title: Lethal giant larvae gene family ( Llgl1 and Llgl2 ) functions as a tumor suppressor in mouse skin epidermis

doi: 10.1101/2023.03.06.531408

Figure Lengend Snippet: A . RNAs extracted from p0 Control ( K14-Cre ) and Llgl1/2 cKO (K14-Cre/ Llgl1/2 fl/fl ) skin epidermises was analyzed by RNA-Seq (3 individuals for each genotype). Significant differences in gene expression (q<0.05) were investigated by Gene Set Enrichment Analysis (GSEA). Significantly enriched Hallmark gene sets in Llgl1/2-/- epidermises are Interferon gamma and TNFA signaling via NFkB sets (q=0.01 and q=0.009). B . Western blot analyses of proteins extracted from epidermises of newborn mice with indicated genotypes. Note increase in phosphorylation of IKKbeta, RelA and aPKC in Llgl1/2 -/- cKO and Llgl1/2 -/- /Trp53 +/- cKO, but not in Trp53 +/- cKO epidermises. * Indicates non-specific background protein band.

Article Snippet: For Western blotting we used the following antibodies: rabbit anti-LLGL1 (1:20,000, Sigma, HPA023569); rabbit anti-LLGL2 (1:1000, Sigma, HPA022913); rabbit anti-RelA-p65 (1:1000, Cell Signaling, #3033); rabbit anti-p-RelA-p65 (1:1000, Cell Signaling, #8242); mouse anti-aPKC (1:1000, Santa Cruz, sc-17781); rabbit anti-p-IKKα/β (1:500, Cell Signaling, #2697), rabbit anti-IKKβ (1:1000, Cell Signaling, #8943), rabbit anti-p-YAP (1:500, Cell Signaling, #4911), rabbit anti-YAP (1:500, Cell Signaling, #4912), rabbit anti-LATS1/2 (1:1000, AbCam, #ab70565), rabbit anti-p-LATS1/2 (1:500, Cell Signaling, #9159), mouse anti-p-AKT (1:1000, Cell Signaling, #4051), rabbit anti-AKT (1:1000, Cell Signaling, #9272), mouse anti-P-MAPK (1:2000, Sigma #M-8159), rabbit anti-MAPK (1:500, Sigma #M-5670), rabbit anti-TAZ, (1:1000, Sigma, #HPA007415), mouse anti-beta actin (1:5000, Sigma, #A5441).

Techniques: Control, RNA Sequencing, Gene Expression, Western Blot, Phospho-proteomics

Journal: bioRxiv

Article Title: Lethal giant larvae gene family ( Llgl1 and Llgl2 ) functions as a tumor suppressor in mouse skin epidermis

doi: 10.1101/2023.03.06.531408

Figure Lengend Snippet: A . Knockdown efficiency of lentiviral shRelA constructs. Keratinocytes were stably transduced with shCtrl, shRelA#1 and shRelA#2 lentiviruses and analyzed by Western blotting with anti-RelA and anti-beta-actin antibodies. B . Quantitation of one hour pulse EdU incorporation of primary keratinocytes with indicated genotypes stably transduced with indicated shRNA lentiviruses. C . Knockout efficiency of RelA targeting CRISPR lentiviral constructs. Keratinocytes were stably transduced with sgCtrl, sgRelA#1 and sgRelA#2 lentiviruses and analyzed by Western blotting with anti-RelA and anti-beta-actin antibodies. D . Quantitation of one hour pulse EdU incorporation of primary keratinocytes with indicated genotypes stably transduced with indicated sgRNA lentiviruses. Graphs show mean values with standard deviation. ** - indicates P value <0.01. *** - indicates P value <0.001. P value was determined using Student’s t-test.

Article Snippet: For Western blotting we used the following antibodies: rabbit anti-LLGL1 (1:20,000, Sigma, HPA023569); rabbit anti-LLGL2 (1:1000, Sigma, HPA022913); rabbit anti-RelA-p65 (1:1000, Cell Signaling, #3033); rabbit anti-p-RelA-p65 (1:1000, Cell Signaling, #8242); mouse anti-aPKC (1:1000, Santa Cruz, sc-17781); rabbit anti-p-IKKα/β (1:500, Cell Signaling, #2697), rabbit anti-IKKβ (1:1000, Cell Signaling, #8943), rabbit anti-p-YAP (1:500, Cell Signaling, #4911), rabbit anti-YAP (1:500, Cell Signaling, #4912), rabbit anti-LATS1/2 (1:1000, AbCam, #ab70565), rabbit anti-p-LATS1/2 (1:500, Cell Signaling, #9159), mouse anti-p-AKT (1:1000, Cell Signaling, #4051), rabbit anti-AKT (1:1000, Cell Signaling, #9272), mouse anti-P-MAPK (1:2000, Sigma #M-8159), rabbit anti-MAPK (1:500, Sigma #M-5670), rabbit anti-TAZ, (1:1000, Sigma, #HPA007415), mouse anti-beta actin (1:5000, Sigma, #A5441).

Techniques: Knockdown, Construct, Stable Transfection, Transduction, Western Blot, Quantitation Assay, shRNA, Knock-Out, CRISPR, Standard Deviation

Journal: bioRxiv

Article Title: Lethal giant larvae gene family ( Llgl1 and Llgl2 ) functions as a tumor suppressor in mouse skin epidermis

doi: 10.1101/2023.03.06.531408

Figure Lengend Snippet: A . Knockdown efficiency of lentiviral sh-aPKC constructs. Keratinocytes were stably transduced with shCtrl, sh-aPKC#1 and sh-aPKC#2 lentiviruses and analyzed by Western blotting with anti-aPKC and anti-beta-actin antibodies. B . Knockdown of aPKC erases the differences in IKK-beta activity between Llgl1/2 -/- /Trp53 +/- and Llgl1/2 fl/fl /Trp53 +/fl controls. Keratinocytes were stably transduced with shCtrl and sh-aPKC#1 lentiviruses and analyzed by Western blotting with anti-LLGL1/2, anti-aPKC, anti-phospho-IKKbeta (P-IKKbeta), anti-total IKKbeta and anti-beta-actin antibodies. C . Knockdown of aPKC erases the differences in activating RelA phosphorylation between Llgl1/2 -/- /Trp53 +/- and Llgl1/2 fl/fl /Trp53 +/fl controls. Keratinocytes were stably transduced with shCtrl and sh-aPKC#1 lentiviruses and analyzed by Western blotting with anti-aPKC, anti-phospho-RelA (P-RelA), anti-total RelA and anti-beta-actin antibodies. D . Quantitation of pulse EdU incorporation of primary keratinocytes with indicated genotypes stably transduced with indicated shRNA lentiviruses. Graph shows mean values with standard deviation. P value was determined using Student’s t-test. E . Hypothetical model of NFkB signaling activation in Llgl1/2 -/- keratinocytes.

Article Snippet: For Western blotting we used the following antibodies: rabbit anti-LLGL1 (1:20,000, Sigma, HPA023569); rabbit anti-LLGL2 (1:1000, Sigma, HPA022913); rabbit anti-RelA-p65 (1:1000, Cell Signaling, #3033); rabbit anti-p-RelA-p65 (1:1000, Cell Signaling, #8242); mouse anti-aPKC (1:1000, Santa Cruz, sc-17781); rabbit anti-p-IKKα/β (1:500, Cell Signaling, #2697), rabbit anti-IKKβ (1:1000, Cell Signaling, #8943), rabbit anti-p-YAP (1:500, Cell Signaling, #4911), rabbit anti-YAP (1:500, Cell Signaling, #4912), rabbit anti-LATS1/2 (1:1000, AbCam, #ab70565), rabbit anti-p-LATS1/2 (1:500, Cell Signaling, #9159), mouse anti-p-AKT (1:1000, Cell Signaling, #4051), rabbit anti-AKT (1:1000, Cell Signaling, #9272), mouse anti-P-MAPK (1:2000, Sigma #M-8159), rabbit anti-MAPK (1:500, Sigma #M-5670), rabbit anti-TAZ, (1:1000, Sigma, #HPA007415), mouse anti-beta actin (1:5000, Sigma, #A5441).

Techniques: Knockdown, Construct, Stable Transfection, Transduction, Western Blot, Activity Assay, Phospho-proteomics, Quantitation Assay, shRNA, Standard Deviation, Activation Assay

Journal: Frontiers in Pharmacology

Article Title: Novel mitophagy inducer TJ0113 alleviates pulmonary inflammation during acute lung injury

doi: 10.3389/fphar.2025.1590458

Figure Lengend Snippet: TJ0113 attenuates inflammation by inhibiting NF-κB activation. (A) BMDMs were pretreated with TJ0113 (10 μM, 6 h), followed by stimulation with LPS (100 ng/mL, 5 min). Western blotting assessed the expression of p-IKK, IKK-β, IκB, p-IκB, p65, and p-p65. (B–E) BMDMs were co-treated with TJ0113 (10 μM) and LPS (100 ng/mL) for 6 h. The mRNA expression level of Il-6 , Cxcl1 , Cxcl2 , and Tnf-α and corresponding cytokine concentrations of cell culture supernatant. Data are presented as mean ± SEM, n=3. ns = no significance; **, P<0.01; ***, P<0.001; ****, P<0.0001. Ordinary two-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: The primary antibodies used include: LC3B rabbit polyclonal antibody (Abcam, ab168831), β-actin mouse monoclonal antibody (Cell Signaling Technology, 3700), P-IKK α/β rabbit monoclonal antibody (Cell Signaling Technology, 2697), P-IκBα rabbit monoclonal antibody (Cell Signaling Technology, 2859), p-p65 rabbit monoclonal antibody (Cell Signaling Technology, 3033), p65 rabbit monoclonal antibody (Cell Signaling Technology, 8242), IκBα rabbit polyclonal antibody (Cell Signaling Technology, 9242), p62 rabbit polyclonal antibody (Cell Signaling Technology, 5114), IKKα/β rabbit monoclonal antibody (Cell Signaling Technology, 2697), COXIV rabbit polyclonal antibody (Cell Signaling Technology, 4850), TOM20 mouse monoclonal antibody (Santa Cruz, sc-17764), p-PINK1 rabbit polyclonal antibody (Signalway Antibody, 29297), NLRP3 mouse monoclonal antibody (AdipoGen, AG-20B-0014), Total and Cleaved IL-1β Antibody (Abmart, P50520-1R1S), and caspase-1 rabbit polyclonal antibody (Proteintech, 22915-1-AP).

Techniques: Activation Assay, Western Blot, Expressing, Cell Culture

Journal: Cardiology Research and Practice

Article Title: Upregulation of MicroRNA-125b Leads to the Resistance to Inflammatory Injury in Endothelial Progenitor Cells

doi: 10.1155/2020/6210847

Figure Lengend Snippet: Upregulation of miR-125b in EPCs attenuated the expression of proinflammatory factors and ameliorated the TNF- α -induced cell apoptosis. EPCs were treated with TNF- α (10 ng/mL) for 1 h after transfection. The mRNA levels of proinflammatory factors (TNF- α , IL-1 β , and IL-6) were measured by qRT-PCR (a). Cell apoptosis was determined by flow cytometry using annexin V staining (b). The activation of caspase3 was analyzed by western blotting. Representative (c) and quantification (d) of caspase3 level (normalized to β -actin). The activation of NF- κ B was determined by the level of p-p65 in EPCs using western blotting. Representative (e) and quantification (f) of p-p65 level (normalized to p65) ( ∗ p < 0.05 vs. NC mimic with TNF- α treatment, n = 5).

Article Snippet: Proteins were extracted with cell lysis buffer (Cell Signaling Technology) and analyzed with by western blotting by using p-NF κ B p65 antibody (Ser 276) (1 : 1000, Santa Cruz, sc-101749), NF- κ B p65 (1 : 1000, Cell Signaling, 8242T) and rabbit anti- β -actin antibodies (1 : 3000; Cell Signaling Technology).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Flow Cytometry, Staining, Activation Assay, Western Blot

Journal: International immunology

Article Title: Increased urinary exosomal SYT17 levels in chronic active antibody-mediated rejection after kidney transplantation via the IL-6 amplifier.

doi: 10.1093/intimm/dxaa032

Figure Lengend Snippet: Fig. 2. NFκB and STAT3 activation in tubular cells of CAAMR patients. (A) Immunohistochemistry of phosphorylated NFκB p65 (p-p65) and phosphorylated STAT3 (p-STAT3) using biopsy specimens from KTx patients with NED, IF/TA, CNI-T or CAAMR. Representative images of a single patient sample are shown for each group. Scale bars are 100 μm. Red arrows are examples of p-p65 and p-STAT3 double-positive cells. (B) Immunohistochemistry of co-staining with p-p65 (brown) and SYT17 (red), or p-STAT3 (brown) and SYT17 (red) in the tubular and glomeruli regions of specimens from a CAAMR patient. Examples of SYT17-expressing cells with phosphorylated p65 and STAT3 are indicated with red arrows. Representative images are shown. Scale bars are 100 μm.

Article Snippet: For double staining of SYT17 with STAT3 pY705 or with phospho NFκB p65 Ser276, the Polink DS-RR-Hu/Ms A Kit (GBI Labs, Bothell, WA, USA) was used with a slight modification.

Techniques: Activation Assay, Immunohistochemistry, Staining, Expressing

Journal: Molecular Oncology

Article Title: WBP2 promotes BTRC mRNA stability to drive migration and invasion in triple‐negative breast cancer via NF‐κB activation

doi: 10.1002/1878-0261.13048

Figure Lengend Snippet: WBP2 activates TNF‐α‐induced NF‐κB activity in TNBC cells. (A) WBP2 positively regulates tumor necrosis factor alpha (TNF‐α)‐induced nuclear factor of kappa beta (NF‐κB) transcriptional activity in MDA‐MB‐231, MDA‐MB‐468, BT549 (i) NF‐κB reporter assay performed in three TNBC cell lines. Cells were harvested after 6h of TNF‐α stimulation. Luciferase signals representing NF‐κB reporter activity were normalized with promoter Renilla signals and quantified related to scrambled (scr) siRNA/vector control cells unstimulated with TNF‐α. (ii) Immunoblot to validate WBP2 knockdown or overexpression. The data are represented as the mean ± SEM of three independent experiments. * P < 0.05, ** P < 0.01 (one‐way ANOVA followed by post hoc Bonferroni test). (B) WBP2 silencing reduced TNF‐α‐stimulated nuclear accumulation of NF‐κB p65 subunit in MDA‐MB‐231 cells. (i) Representative immunofluorescence (IF) images showing p65 (red) localization upon knockdown of WBP2 and 30min of TNF‐α stimulation in MDA‐MB‐231 cells. O represents cells that are not treated with TNF‐α. Pooled WBP2 siRNAs (siWBP2 #1 + 2) were generated by combining siWBP2 #1 and siWBP2 #2 in equal amount. Nuclei were stained with Hoechst (blue). Scale bar (white; bottom right), 100µm. White arrow indicates examples of cells with p65 localized in the nucleus. (ii) The nuclear:cytoplasmic ratio of p65 was quantified using imagej software. The data are represented as the mean ± SEM, n = 3. * P < 0.05, ** P < 0.01 (one‐way ANOVA followed by post hoc Bonferroni test). (iii) Immunoblot to confirm efficiency of WBP2 knockdown and determine total cellular p65 expression. β‐Tubulin was used as the loading control. (C) (i) Representative immunoblot of subcellular fractionation experiment showing WBP2 and p65 nuclear and cytoplasmic expression in MDA‐MB‐231 cells after 30 min of TNF‐α induction. (ii&iii) Densitometry analysis of (ii) nuclear WBP2 and (iii) p65 signal was performed to confirm the nuclear expression after TNF‐α stimulation. The data are represented as the mean ± SEM, n = 3, n = 3. N.S, nonsignificant, ** P < 0.01 (unpaired t ‐test).

Article Snippet: Anti‐IKKα rabbit, anti‐IKKβ rabbit, anti‐Phospho‐IKKα/β (Ser176/180) rabbit, anti‐IκBα mouse, anti‐Phospho‐IκBα (Ser32/36) mouse, anti‐NF‐κB p65 rabbit, anti‐NF‐κB p65 mouse, and anti‐Phospho‐NFκB p65 (Ser536) rabbit monoclonal antibodies were obtained from Cell Signaling Technology Inc. (Danvers, MA, USA).

Techniques: Activity Assay, Reporter Assay, Luciferase, Plasmid Preparation, Control, Western Blot, Knockdown, Over Expression, Immunofluorescence, Generated, Staining, Software, Expressing, Fractionation

Journal: Frontiers in Pharmacology

Article Title: Vitamin D 3 preserves blood retinal barrier integrity in an in vitro model of diabetic retinopathy

doi: 10.3389/fphar.2022.971164

Figure Lengend Snippet: Effect of vitamin D 3 on NF-κB activation in HG-challenged HRECs. (A) Representative images of phosphorylated p-NFκB p65 (red) translocation into the nuclei (blue) stained with DAPI. HRECs were pre-treated with vitamin D 3 (1 µM) for 24 h and then with or without high glucose for 24 h. (B) Fluorescence semi-quantification of p-NFκB p65 protein (mean grey levels) into the nucleus. Nuclei were labeled with DAPI (blue). Images were acquired at × 40 magnification. Scale bar: 10 µm. Values are reported as mean ± SD; n = 4. Data were analyzed by one-way ANOVA and Tukey post-hoc test for multiple comparisons. * p < 0.05 vs. control; † p < 0.05 vs. HG.

Article Snippet: After blocking with 5% NGS and 0.3% Triton X-100 in PBS solution, for 30 min at room temperature, the cells were incubated with the mouse-anti- phospho-NFκB p65 (Ser536; 1:200, Catalog n. 3036 Cell signaling, Technology, Danvers, MA, United States ) in 1% NGS and 0.2% Triton X-100 in PBS solution overnight at 4°C.

Techniques: Activation Assay, Translocation Assay, Staining, Fluorescence, Labeling, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Reactive oxygen species level defines two functionally distinctive stages of inflammatory dendritic cell development from mouse bone marrow.

doi: 10.4049/jimmunol.0903458

Figure Lengend Snippet: FIGURE 3. ROShi DCs demonstrate altered NF-kB p65 or p38 MAPK expression and phosphorylation profiles in the steady-state or activated state. A, Cells from day 3 or day 6 GM-CSF culture were labeled with anti- CD11c-APC, fixed, and permeabilized. Cells were washed and labeled with FITC-conjugated mAb NF-kB p65 or with p38 (H-147) rabbit polyclonal IgG, followed by secondary FITC-conjugated sheep anti-rabbit Ig. The histograms compare NF-kB p65 (left) and p38 (right) expression between day 3 (dotted line) and day 6 (solid line). The shaded area for the NF-kB p65 histogram represents nonstained cells, and for p38 detection it represents the secondary Ab alone. B–D, To detect activation of NF-kB p65 or p38 MAPK, cells prestimulated with LPS plus zymosan or LPS alone were permeabilized, labeled with anti-CD11c–APC, and incubated with the p-NF-kB p65 (Ser276) or p-p38 MAPK (Thr180/Tyr182) mouse Ab, followed by labeling with the corresponding FTIC-conjugated secondary Ab. The phosphorylated subunit of the kinase was detected and analyzed in the histogram, by which the true level of phosphorylation was calculated, as described in Materials and Methods. The shaded area represents the cells stained with the secondary Ab alone. The steady-state level of p- NF-kB p65 (Ser276) or p-p38 MAPK (Thr180/Tyr182) (phospho-level) of day 3 and day 6 DCs was compared (B) and is shown as a dotted line in the histogram (C), which also shows the activated level after TLR stimulation (solid line) at 90 min for NF-kB p65 (Ser276) and at 15 min for p38 MAPK (Thr180/Tyr182). The increase in phosphorylation level (phospho-increase) was determined at specific time points along stimulation and its kinetics was demonstrated (D). Data shown are representative of at least three experiments. p, phosphorylated.

Article Snippet: DCs were permeabilized and incubated with p-NF-kB p65 (Ser276) rabbit polyclonal IgG (Santa CruzBiotechnology), followed by FITC-conjugated sheep anti-rabbit Ig stain.

Techniques: Expressing, Phospho-proteomics, Labeling, Activation Assay, Incubation, Staining