Journal: International journal of molecular sciences

Article Title: WGA-M001, a Mixture of Total Extracts of Tagetes erecta and Ocimum basilicum , Synergistically Alleviates Cartilage Destruction by Inhibiting ERK and NF-κB Signaling.

doi: 10.3390/ijms242417459

Figure Lengend Snippet: Figure 4. WGA-M001 regulated OA-related molecules through dephosphorylation in the NF-κB and ERK pathways. For in silico analysis, 200 µg/mL of Tagetes erecta, Ocimum basilicum, or WGA-M001 was used for treatment of chondrocytes for 12 h along with IL-1β (1 ng/mL), and RNA sequencing was performed. (A) In each signaling pathway, the number of genes upregulated by IL-1β and then downregulated by T. erecta, O. basilicum, or WGA-M001 was shown. (B,C) Protein levels of pp38, p38, pJNK, JNK, pERK, ERK, pp65, p65, and IκB were detected by Western blot analysis and relative intensities were quantified by densitometry (n = 5). Each protein level was normalized to ERK. Data are represented as mean ± SD as results of analysis by one-way ANOVA with Dunnett’s multiple comparisons test (n = 5). * p < 0.05, **** p < 0.0001, and n.s = not significant.

Article Snippet: 2023, 24, 17459 12 of 16 cam), rabbit anti-COX-2 (ab52237; Abcam), mouse anti-IκB (9242; Cell Signaling Technology (CST), Danvers, MA, USA), mouse anti-p65 (#6956; CST), mouse anti-pp65 (#13346; CST), mouse anti-p38 (#9212; CST), mouse anti-pp38 (#9215S; CST), mouse anti-c-JNK (#9252S; CST), mouse anti-pJNK (#9251S; CST), and mouse anti-pERK (#9101S; CST).

Techniques: De-Phosphorylation Assay, In Silico, RNA Sequencing, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: LncRNA NEAT1 promotes apoptosis and inflammation in LPS-induced sepsis models by targeting miR-590-3p

doi: 10.3892/etm.2020.9079

Figure Lengend Snippet: Inhibition of NEAT1 suppresses the NF-κB signaling pathway via targeting miR-590-3p. H9c2 cells were transfected with si-NEAT1 and anti-miR-590-3p or their corresponding negative controls (si-NC and anti-NC), followed by treatment with LPS. (A) The protein levels of NF-κB pathway-related factors p-p65 and p65 were detected and (B) quantified using western blot analysis. * P<0.05. NEAT1, nuclear enriched abundant transcript 1; miR, microRNA; si-NEAT1, small interfering RNA targeting NEAT1; LPS, lipopolysaccharide; p, phosphorylated.

Article Snippet: Primary antibodies against Bcl-2 (1:500; cat. no. BA0412), Bax (1:1,000; cat. no. BA0315-2), caspase 3 (1:1,000; cat. no. BA3257), TNF receptor associated factor 6 (TRAF6; 1:1,500; cat. no. A00185), phosphorylated (p)-p65 (1:1,000; cat. no. P00284), p65 (1:2,000; cat. no. A00284) and GAPDH (1:2,000; cat. no. BA2913) were purchased from Wuhan Boster Biological Technology, Ltd. Membranes were then incubated with horseradish peroxidase-labeled secondary antibody (1:5,000; cat no. BA1056; Wuhan Boster Biological Technology, Ltd.) for 1 h at room temperature.

Techniques: Inhibition, Transfection, Western Blot, Small Interfering RNA

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