Journal: Cells

Article Title: Protective Effects of SIRT6 Overexpression against DSS-Induced Colitis in Mice

doi: 10.3390/cells9061513

Figure Lengend Snippet: SIRT6 regulated expression and phosphorylation of TAK1. ( a ) The expression and phosphorylation levels of TAK1 were determined by immunohistochemistry. ( b , c ) The statistical results of ( a ). n = 3–6, ** p < 0.01.

Article Snippet: Sections then were blocked in 5% rat serum and incubated overnight at 4 °C with the diluted biotinylated primary antibody (1:500; rat-anti-mouse; anti-TAK1, #ab109526, Abcam, Cambridge, England, UK; anti-p-TAK1, #4508, Cell Signaling Technology, Danvers, MA, USA; anti-c-Jun, #ab40766, Abcam; anti-p-Jun, #ab32385, Abcam; anti-NF-κB p65 antibody, #ab32536, Abcam; anti-NF-κB p65 (phosphor S536), #ab86299, Abcam; anti-NF-κB p65 (acetyl K310) antibody, #ab19870, Abcam).

Techniques: Expressing, Immunohistochemistry

Journal: Artificial cells, nanomedicine, and biotechnology

Article Title: Silence of lncRNA MIAT protects ATDC5 cells against lipopolysaccharides challenge via up-regulating miR-132.

doi: 10.1080/21691401.2019.1626410

Figure Lengend Snippet: Figure 4. Suppressive effect of MIAT silence on the activation of NF-jB and JNK pathways via miR-132. ATDC5 cells were co-transfected with sh-MIAT and miR-132 inhibitor and then subjected to 6 lg/mL LPS. The phosphor/total (p/t) levels of (A,B) p65, IjBa and (C,D) JNK were measured using western blot (n ¼ 3). p < .05 (ANOVA combined with Duncan post-hoc test).

Article Snippet: Anti-caspase-3 (orb378617), anti-cleavedcaspase-3 (orb106556), anti-caspase-9 (orb135175), anticleaved-caspase-9 (orb227889), anti-PARP (orb526607), anti-cleaved-PARP (orb106557), anti-IL-6 (orb6210), anti-IL-8 (orb39299), anti-TNF-a (orb475251), anti-MCP-1 (orb97456), anti-p65 (orb344389), anti-p-p65 (orb14753), anti-IjBa (orb338946), anti-p-IjBa (orb99312), anti-JNK (orb38050), anti-p-JNK (orb184488), and anti-b-actin (orb86987) all from Biorbyt (San Francisco, CA).

Techniques: Activation Assay, Transfection, Western Blot

Journal: International Journal of Cardiology. Heart & Vasculature

Article Title: Macrophage Phospholipase D3 promotes atherosclerosis via exacerbating foam cell formation and inducing inflammatory responses

doi: 10.1016/j.ijcha.2025.101834

Figure Lengend Snippet: PLD3 knockdown attenuates foam cell formation and macrophage inflammation (A) Representative Oil Red O staining images of THP-1 cells transduced with lentiviral shNC or shPLD3, incubated with or without ox-LDL (50 μg/mL) for 24 h. Scale bar: 15 μm. (B) Localization of Dil-ox-LDL in THP-1 cells observed by immunofluorescence microscopy. Dil-ox-LDL (red); Hoechst staining (nuclei, blue). Scale bar: 30 μm. (C) Localization of BODIPY in THP-1 cells observed by immunofluorescence microscopy. BODIPY (green); Hoechst staining (nuclei, blue). Scale bar: 200 μm. (D and H-J) Western blot analysis of SR-A, CD36 and LOX-1 protein expression in THP-1 cells. GAPDH served as loading control (n = 3). (E and K) Western blot analysis of p-p65 and p65 protein expression in THP-1 cells. GAPDH served as loading control (n = 3). (F and G) mRNA levels of IL-1β and TNF-α in THP-1 cells determined by RT-qPCR. GAPDH served as internal control (n = 3).

Article Snippet: p-p65 , Abmart , TP56372 , 1:1000.

Techniques: Knockdown, Staining, Transduction, Incubation, Immunofluorescence, Microscopy, Western Blot, Expressing, Control, Quantitative RT-PCR

Journal: Journal of dairy science

Article Title: Thiamine ameliorates inflammation of the ruminal epithelium of Saanen goats suffering from subacute ruminal acidosis.

doi: 10.3168/jds.2019-16944

Figure Lengend Snippet: Figure 2. Effects of thiamine supplementation on the relative protein (A) IL-1β, (B) IL-6, (C) tumor necrosis factor α (TNF-α), (D) p65, and (E) p-p65 expression levels in the ruminal epithelium of dairy goats with SARA. The protein expression value equals densitometry units of selected protein/densitometry units of β-actin detected by Western blotting. Values are presented as means ± SEM; n = 6 goats/group. Means within each protein with different letters (a–c) are significantly different (P < 0.05). CON = control; HC = high-concentrate diet; THC = high- concentrate diet supplemented with 200 mg of thiamine/kg of DMI.

Article Snippet: These membranes were blocked with 5% skim milk (5% BSA for phosphorylated p65) at 25°C for 2 h, followed by an incubation with primary antibodies, including anti-p65 (1:300, sc-109; Santa Cruz Biotechnology Inc., Dallas, TX), anti-p p65 (Ser 536; 1:300, sc-33020; Santa Cruz Biotechnology Inc.), anti-IL-1β (1:300, sc-7884; Santa Cruz Biotechnology Inc.), anti-TNF-α (1:300, sc-8301; Santa Cruz Biotechnology Inc.), anti-IL-6 (1:300, sc- 1265; Santa Cruz Biotechnology Inc.), and anti-β-actin (1:10,000, BS6007M; Bioworld Technology Inc., St. Louis Park, MN) at 4°C overnight.

Techniques: Expressing, Western Blot, Control

Journal: Frontiers in Oncology

Article Title: SPNS2 Downregulation Induces EMT and Promotes Colorectal Cancer Metastasis via Activating AKT Signaling Pathway

doi: 10.3389/fonc.2021.682773

Figure Lengend Snippet: Effects of SPNS2 on the AKT signaling pathway. (A) GSEA analysis in CCLE CRC cell lines showed that SPNS2 expression negatively correlated with “HALLMARK PI3K AKT MTOR SIGNALING” and “HALLMARK MTORC1 SIGNALING”. (B, C) The relative activities (mean ± S.D.) of AKT, ERK, STAT3, and NF-κB pathways were determined via Qiagen reporter luciferase assay in SPNS2-overexpressing HCT116 cells or SPNS2 knockdown SW480 cells versus the NC counterparts. **p < 0.01 (D) A representative western blot of total AKT, phosphorylated AKT (Ser473), ERK, phosphorylated ERK, P65 and phosphorylated P65 in the indicated CRC cells. (E) MK2206 reduced the phosphorylation level of AKT in SW480 si-SPNS2 cells and SC79 enhanced the phosphorylation level of AKT in HCT116 OE-SPNS2 cells. (F) MK2206 reduced cell migration and invasion in SW480 si-SPNS2 cells (scale bar = 100 µm). (G) SC79 enhanced cell migration and invasion in HCT116 OE-SPNS2 cells (scale bar = 100 µm).

Article Snippet: Primary antibodies used in the present study are as follow: SPNS2(1:500, Novus #NBP1-54345), p-AKT(1:1000, CST #4060), AKT (1:1000, CST #C6717), p-ERK1/2 (1:1000, CST #4370), ERK1/2 (1:1000, CST #4695), p-p65 (1:1000, Immunoway #YD0191), p65 (1:1000, proteintech #10745-1-AP), p-JNK (1:1000, CST #9255), JNK (1:1000, CST #9252), and GAPDH (1:2000, proteintech # 60004-1-Ig).

Techniques: Expressing, Luciferase, Knockdown, Western Blot, Phospho-proteomics, Migration

Journal: Cancer Science

Article Title: Tumor exosomal RNPEP promotes lung metastasis of liver cancer via inducing cancer‐associated fibroblast activation

doi: 10.1111/cas.16417

Figure Lengend Snippet: Antibodies used in western blotting.

Article Snippet: p‐NF‐κB p65 , 1:1000 , AP0124 , Abclonal.

Techniques: Western Blot

Journal: Journal of Biomedicine and Biotechnology

Article Title: Hunting for Serine 276-Phosphorylated p65

doi: 10.1155/2010/275892

Figure Lengend Snippet: Anti-phospho-p65 Ser276 (Cell Signaling no. 3037) detects atypical (with MWs of 80 and 130 kDa), inducible immunoreactivities in different cell types, induced with different NF- κ B-activating agents. Cells were stimulated for increasing times (L929sA, A549, L363) or for 30 min (Raw264.7, C2C12, 1321N1) with TNF- α (2000 IU/mL), LPS (1 μ g/mL), PMA (10 ng/mL), forskolin (forsk, 10 μ M) or isoproterenol (iso, 10 μ M). For blocking experiments, lysates were loaded on one gel in duplicate and after transfer, blots were cut in half. Immunodetection was performed in parallel, using Cell Signaling no. 3037 or Cell Signaling no. 3037 preincubated for at least 30 minutes with double volume of blocking peptide (BLOCK). Arrow 1 indicates an immunoreactive band with an MW of 130 kDa; arrow 2 indicates an immunoreactive band of 80 kDa.

Article Snippet: Four rabbit polyclonal antibodies directed against the phosphorylated Ser276 residue of p65 were used in this study: anti-P-p65 Ser276 (no. 3037) from Cell Signaling (Danvers, MA), anti-P-p65 Ser276 (no. 11011) from Signalway Antibody (SAB, Pearland, TX), anti-P-p65 Ser276 (no. 100-401-264) from Rockland (Gilbertsville, PA), and a homemade anti-P-p65 Ser276 antibody (kind gift of Prof. Dr. P. Cohen, University of Dundee, UK; produced and purified by Drs. C. Armstrong and J. Leitch).

Techniques: Blocking Assay, Immunodetection

Journal: Journal of Biomedicine and Biotechnology

Article Title: Hunting for Serine 276-Phosphorylated p65

doi: 10.1155/2010/275892

Figure Lengend Snippet: The Cell Signaling no. 3037 anti-P-p65 Ser276-immunoreactive band persists in cells where p65 is silenced via an siRNA approach. 1321N1 cells were transiently transfected with control or p65 siRNA as described in . Cells were induced for 90 min with TNF- α (2000 IU/mL), iso (10 μ M), or a combination of both stimuli. Presence of Ser276-phosphorylated p65 in cell lysates was analysed by Western Blot using Cell Signaling no. 3037 (A1). 130 and 80 kDa immunoreactivities are indicated by arrows. Western Blot to detect anti-P-p65Ser536 (65 kDa arrow) was performed on the same lysates (B1). Blots A1 and B1 were extensively washed and reprobed with anti-p65 to investigate efficiency of p65 knock-down. Anti-p65 detected bands are marked with arrows (A2, B2). Finally, blots were reprobed with anti-tubulin to check loading efficiency. Anti-tubulin-detected bands are marked with arrows (A3, B3).

Article Snippet: Four rabbit polyclonal antibodies directed against the phosphorylated Ser276 residue of p65 were used in this study: anti-P-p65 Ser276 (no. 3037) from Cell Signaling (Danvers, MA), anti-P-p65 Ser276 (no. 11011) from Signalway Antibody (SAB, Pearland, TX), anti-P-p65 Ser276 (no. 100-401-264) from Rockland (Gilbertsville, PA), and a homemade anti-P-p65 Ser276 antibody (kind gift of Prof. Dr. P. Cohen, University of Dundee, UK; produced and purified by Drs. C. Armstrong and J. Leitch).

Techniques: Transfection, Control, Western Blot, Knockdown

Journal: Journal of Biomedicine and Biotechnology

Article Title: Hunting for Serine 276-Phosphorylated p65

doi: 10.1155/2010/275892

Figure Lengend Snippet: Four independent anti-P-p65 Ser276 antibodies detect inducible bands, with MWs of 80 and 130 kDa, that do not disappear upon p65 knock-down, but are inhibited when PKAc α is silenced. 1321N1 cells were transiently transfected with control, p65 or PKAc α siRNA, as described in . Cells were induced for 90 min with TNF- α (2000 IU/mL), iso (10 μ M) or a combination of both stimuli. Presence of Ser276-phosphorylated p65 in cell lysates was analysed by Western Blot using SAB no. 11011 (A1), Rockland no. 100-401-264 (B1), Cell Signaling no. 3037 (B3), or a homemade anti-P-p65 Ser276 antibody (C1). 130 and 80 kDa immunoreactivities are indicated by arrows (1 and 2, resp.). Blots were extensively washed and reprobed with a mixture of anti-p65 and anti-PKAc α to investigate knock-down efficiency. Anti-p65-detected bands are marked by the upper arrow, anti-PKAc α -reactive bands are marked by the lower arrow (A2, B2, C2). Tubulin loading controls are added as Supplementary Figure 1.

Article Snippet: Four rabbit polyclonal antibodies directed against the phosphorylated Ser276 residue of p65 were used in this study: anti-P-p65 Ser276 (no. 3037) from Cell Signaling (Danvers, MA), anti-P-p65 Ser276 (no. 11011) from Signalway Antibody (SAB, Pearland, TX), anti-P-p65 Ser276 (no. 100-401-264) from Rockland (Gilbertsville, PA), and a homemade anti-P-p65 Ser276 antibody (kind gift of Prof. Dr. P. Cohen, University of Dundee, UK; produced and purified by Drs. C. Armstrong and J. Leitch).

Techniques: Knockdown, Transfection, Control, Western Blot

Journal: Journal of Biomedicine and Biotechnology

Article Title: Hunting for Serine 276-Phosphorylated p65

doi: 10.1155/2010/275892

Figure Lengend Snippet: Presence of anti-P-p65 Ser276-reactive bands in p65 deficient MEF cells. p65 knock-out MEF cells (−/−), or p65 −/− MEF cells reconstituted with wild type p65 (p65 wt) or p65 with Ser276 mutated to alanine (p65 S/A), were induced with forskolin (forsk, 10 μ M) for 0, 30, and 60 min. Presence of Ser276-phosphorylated p65 in cell lysates was analysed by Western Blot using Cell Signaling no. 3037 (A1) or SAB no. 11011 (B1). Blots were extensively washed and reprobed with anti-p65 to confirm p65 absence in −/− MEFs, and p65 presence in wt and S/A reconstituted MEFs (A2 and B2, arrows indicate anti-p65 detected bands).

Article Snippet: Four rabbit polyclonal antibodies directed against the phosphorylated Ser276 residue of p65 were used in this study: anti-P-p65 Ser276 (no. 3037) from Cell Signaling (Danvers, MA), anti-P-p65 Ser276 (no. 11011) from Signalway Antibody (SAB, Pearland, TX), anti-P-p65 Ser276 (no. 100-401-264) from Rockland (Gilbertsville, PA), and a homemade anti-P-p65 Ser276 antibody (kind gift of Prof. Dr. P. Cohen, University of Dundee, UK; produced and purified by Drs. C. Armstrong and J. Leitch).

Techniques: Knock-Out, Western Blot

Journal: Journal of Biomedicine and Biotechnology

Article Title: Hunting for Serine 276-Phosphorylated p65

doi: 10.1155/2010/275892

Figure Lengend Snippet: The detected 130 kDa and 80 kDa immunoreactivities are not NF- κ B p105/p50 or c-Rel. 1321N1 cells were transiently transfected with control, p105/p50 or c-Rel siRNA as described in . Cells were induced for 30 min with TNF- α (2000 IU/mL) or iso (10 μ M). Presence of Ser276-phosphorylated p65 in cell lysates was analysed by Western Blot using Cell Signaling no. 3037 (A1, B1). 130 and 80 kDa immunoreactivities are indicated by arrows (1 and 2 resp.). Because of the overlap of the 130 kDa and the p105 immunoreactive bands, blot A was first stripped and reprobed with anti-p105/p50 (A2). p105 immunoreactivity is indicated by the upper arrow; p50 immunoreactivity is indicated by the lower arrow. Blot B was extensively washed and reprobed (without stripping) with anti-c-Rel (B2). c-Rel immunoreactivity is indicated by an arrow. Tubulin was used as a loading control (A3, B3).

Article Snippet: Four rabbit polyclonal antibodies directed against the phosphorylated Ser276 residue of p65 were used in this study: anti-P-p65 Ser276 (no. 3037) from Cell Signaling (Danvers, MA), anti-P-p65 Ser276 (no. 11011) from Signalway Antibody (SAB, Pearland, TX), anti-P-p65 Ser276 (no. 100-401-264) from Rockland (Gilbertsville, PA), and a homemade anti-P-p65 Ser276 antibody (kind gift of Prof. Dr. P. Cohen, University of Dundee, UK; produced and purified by Drs. C. Armstrong and J. Leitch).

Techniques: Transfection, Control, Western Blot, Stripping Membranes

Journal: Journal of Biomedicine and Biotechnology

Article Title: Hunting for Serine 276-Phosphorylated p65

doi: 10.1155/2010/275892

Figure Lengend Snippet: In vitro recognition of the phosphorylated Serine 276 residue of p65 by Cell Signaling no. 3037. Recombinant wt p65-GST or SC mutant p65-GST fusion proteins were in vitro phosphorylated using recombinant MSK-1. 250 ng of the recombinant proteins were spotted on a nitrocellulose membrane and subjected to Western Blotting with Ser276 phospho-specific p65 antibody, either preincubated with 10 μ g/mL phosphorylated peptide that was used to generate the antibody (upper panel) or not. 1 μ g of recombinant protein was separated by SDS-PAGE after the in vitro kinase assays performed either in the presence of active MSK-1 and/or 10 μ M H89 (lower panel) or not.

Article Snippet: Four rabbit polyclonal antibodies directed against the phosphorylated Ser276 residue of p65 were used in this study: anti-P-p65 Ser276 (no. 3037) from Cell Signaling (Danvers, MA), anti-P-p65 Ser276 (no. 11011) from Signalway Antibody (SAB, Pearland, TX), anti-P-p65 Ser276 (no. 100-401-264) from Rockland (Gilbertsville, PA), and a homemade anti-P-p65 Ser276 antibody (kind gift of Prof. Dr. P. Cohen, University of Dundee, UK; produced and purified by Drs. C. Armstrong and J. Leitch).

Techniques: In Vitro, Residue, Recombinant, Mutagenesis, Membrane, Western Blot, SDS Page