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: To investigate whether other available antibodies would perhaps be more useful to detect Ser276 phosphorylated p65 via Western Blotting, we investigated phosphorylation of p65 at Ser276 using two anti-P-p65 Ser276 antibodies distributed by independent companies (no. 11011 from SAB and no. 100-401-264 from Rockland).

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: To investigate whether other available antibodies would perhaps be more useful to detect Ser276 phosphorylated p65 via Western Blotting, we investigated phosphorylation of p65 at Ser276 using two anti-P-p65 Ser276 antibodies distributed by independent companies (no. 11011 from SAB and no. 100-401-264 from Rockland).

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: To investigate whether other available antibodies would perhaps be more useful to detect Ser276 phosphorylated p65 via Western Blotting, we investigated phosphorylation of p65 at Ser276 using two anti-P-p65 Ser276 antibodies distributed by independent companies (no. 11011 from SAB and no. 100-401-264 from Rockland).

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: To investigate whether other available antibodies would perhaps be more useful to detect Ser276 phosphorylated p65 via Western Blotting, we investigated phosphorylation of p65 at Ser276 using two anti-P-p65 Ser276 antibodies distributed by independent companies (no. 11011 from SAB and no. 100-401-264 from Rockland).

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: To investigate whether other available antibodies would perhaps be more useful to detect Ser276 phosphorylated p65 via Western Blotting, we investigated phosphorylation of p65 at Ser276 using two anti-P-p65 Ser276 antibodies distributed by independent companies (no. 11011 from SAB and no. 100-401-264 from Rockland).

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: To investigate whether other available antibodies would perhaps be more useful to detect Ser276 phosphorylated p65 via Western Blotting, we investigated phosphorylation of p65 at Ser276 using two anti-P-p65 Ser276 antibodies distributed by independent companies (no. 11011 from SAB and no. 100-401-264 from Rockland).

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

Journal: Advanced Science

Article Title: Mobile Colistin Resistance Enzyme MCR‐3 Facilitates Bacterial Evasion of Host Phagocytosis

doi: 10.1002/advs.202101336

Figure Lengend Snippet: Compared with the wild‐type strain, mcr‐3 ‐positive AS1 suffered significantly less phagocytosis and showed reduced macrophage stimulation. A) Representative confocal laser scanning microscopy images showing phagocytosis of AS1 strains containing plasmids pHSG299 or pHSG299‐ mcr‐3 , along with uninfected controls. Calculation of the phagocytosis index (engulfed bacteria per macrophage, calculated from at least five fields per slide) based on confocal microscopy images (≥200 cells were scored per well). B) Representative confocal laser scanning microscopy images showing phagocytosis of DH5 α strains containing plasmids pHSG299 or pHSG299‐ mcr‐3 , along with uninfected controls. Calculation of the phagocytosis index (engulfed bacteria per macrophage, calculated from at least five fields per slide) based on confocal microscopy images (≥200 cells were scored per well). C) Transcription of IL‐1 β and TNF‐ α in RAW264.7 macrophages stimulated with mcr‐3 ‐positive or mcr‐3 ‐negative AS1. D) Transcription of IL‐1 β and TNF‐ α in RAW264.7 macrophages stimulated with MCR‐3‐modified/unmodified LPS extracted from AS1. E) Immunoblotting analysis. Total expression of NF‐ κ B‐pathway proteins (phosphorylated‐p65, p65, β ‐actin, and I κ B α ) in the lysates of RAW264.7 macrophages treated for 60 min with modified/unmodified LPS purified from mcr‐3 ‐positive/negative AS1. F) Nuclear protein expression in RAW264.7 macrophages treated for 60 min with MCR‐3‐modified/unmodified LPS from AS1. Histone was used as a control. Statistical significance was assessed by two‐tailed unpaired t ‐test; ** P < 0.01 and * P < 0.05. Error bars represent means ± SEM from triplicate wells.

Article Snippet: Transferred proteins were detected using specific antibodies against the phosphorylated and nonphosphorylated forms of NF‐ κ B p65, I κ B α , β ‐actin, and Histone H3 (obtained from Beyotime Biotechnology and Cell Signal Technology, Danvers, MA, USA) according to the manufacturer's instructions.

Techniques: Confocal Laser Scanning Microscopy, Confocal Microscopy, Modification, Western Blot, Expressing, Purification, Two Tailed Test

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Guiqi Baizhu Decoction Alleviates Radiation Inflammation in Rats by Modulating the Composition of the Gut Microbiota

doi: 10.1155/2020/9017854

Figure Lengend Snippet: Effect of GQBZD on the inflammatory injury of intestinal in X-ray radiation rats. Histopathological observations of the colon stained with hematoxylin-eosin (a) were carried out under light microscopy with 40x magnification (scale bar 50 μ m). Colon pathology score was counted in microscope (b). Occludin ((c) and (d)), ZO-1 ((c) and (e)), P65 ((f) and (g)), and p-P65 ((f) and (h)) were tested by western blot and in comparison with the X-ray radiation group. Values are presented as mean ± SEM. Differences were assessed by ANOVA for multiple comparisons and denoted as follows: ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ns P > 0.05.

Article Snippet: The membrane was blocked for 1 h in PBST containing 5% milk and subsequently probed with Occludin antibody (GTX114949, GeneTex, Texas, USA), ZO-1 antibody (Ab96587, Cambridge, UK), P65 antibody (YT3108, ImmunoWay, Texas, USA), phosphorylated P65 antibody (B4568, ImmunoWay, Texas, USA), and GAPDH antibody (ab9485, Abcam, Cambridge, UK), respectively, for 2 h. After 1 h incubation with goat-anti-rabbit HRP-conjugated secondary antibody (ab97051, Abcam, Cambridge, UK), the protein bands were detected with luminal reagent (Millipore, Pittsburgh, USA) and their relative intensities were quantified using Image J.

Techniques: Staining, Light Microscopy, Microscopy, Western Blot, Comparison