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

Journal: iScience

Article Title: Mycobacterium tuberculosis specific protein Rv1509 modulates osteoblast and osteoclast differentiation via TLR2 signaling

doi: 10.1016/j.isci.2025.112107

Figure Lengend Snippet: Rv1509 induces osteolysis in mouse skulls (A) 3D reconstruction images of mouse skulls after two weeks of treatment with saline or Rv1509. The data are shown as mean ± SEM. (scale bar: 1 mm; n = 4; ∗, p < 0.05; ∗∗∗, p < 0.001 [Student’s t test]). (B) ELISA results of IL-6 and TNF-α levels in the mouse eye serum. The data are shown as mean ± SEM. ( n = 5; ∗∗, p < 0.01; ∗∗∗, p < 0.001 [Student’s t test]). (C) Representative images of H&E staining, Masson staining, TRAP staining, and immunohistochemical staining for p-p65 in mouse skull tissue(scale bar: 200 μm). (D) Quantitative analysis of TRAP staining, p-p65 immunohistochemical staining, and Masson staining. The data are shown as mean ± SEM. ( n = 3; ∗∗, p < 0.01; ∗∗∗, p < 0.001 [Student’s t test]). (E) Analysis of the calcein double-labeling experiment showing the effects of Rv1509 on bone formation and mineralization, scale bar: 50 μm. (F) Quantitative analysis of the calcein double-labeling experiment including bone mineralization deposit rate (MAR), mineralized surface area (MS/BS), and bone formation rate (BFR) ( n = 3). The data are shown as mean ± SEM. ( n = 3; ∗, p < 0.05; ∗∗∗, p < 0.001 [Student’s t test]).

Article Snippet: Mouse anti-p-p65 Ab , Santa Cruz , Cat#sc-136548; RRID: AB_10610391.

Techniques: Saline, Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemical staining, Labeling

Journal: iScience

Article Title: Mycobacterium tuberculosis specific protein Rv1509 modulates osteoblast and osteoclast differentiation via TLR2 signaling

doi: 10.1016/j.isci.2025.112107

Figure Lengend Snippet:

Article Snippet: Mouse anti-p-p65 Ab , Santa Cruz , Cat#sc-136548; RRID: AB_10610391.

Techniques: Virus, Recombinant, Lysis, Staining, CCK-8 Assay, Enzyme-linked Immunosorbent Assay, BIA-KA, RNA Sequencing, Software

Journal: BMC Molecular and Cell Biology

Article Title: Three functional mutation sites affect the immune response of pigs through altering the expression pattern and IgV domain of the CD4 protein

doi: 10.1186/s12860-020-00333-7

Figure Lengend Snippet: The functional verification of Haplotypes AA and BB at the individual level in pigs. a Functional analysis of differentially expressed genes between genotypes AA and BB in pigs ( n = 10 for AA; n = 16 for BB). b Q-PCR validation of the differentially expressed genes ( n = 6 for AA; n = 6 for BB). β-actin was used as the internal control for Q-PCR. Triplicate samples were analyzed for each treatment, and the results are presented as the mean ± s.e.m. **, P < 0.01. c The protein interaction network of DEGs. The red nodes represented genes that were up-regulated in individuals with genotype BB, and the green nodes represented genes that were down-regulated. The white nodes represented genes that were not significantly different between genotypes AA and BB. d Western blotting detection of the protein levels of CD4 and p-p65 of the two haplotypes (genotype AA, n = 5; genotype BB, n = 5) using anti-CD4 and anti-p65. β-actin was used as internal control

Article Snippet: Membranes were blocked in 5% non-fat milk for 1 h at room temperature and incubated with 1:1000 anti-GFP antibody (ABclonal, AE012), 1:2000 anti-flag antibody (ABclonal, AE005), 1:1000 anti-CD4 antibody (Abcam, UK, 25804), 1:1000 anti-β-actin antibody (Beyotime, Shanghai, China, AF0003), or 1:1000 anti-p-p65 antibody (CST, USA, 4025) at 4 °C overnight.

Techniques: Functional Assay, Biomarker Discovery, Control, Western Blot