phosphorylated p65 (p-p65)  (Santa Cruz Biotechnology)

Journal: Poultry Science

Article Title: Arbutin improves lung injury in chicks induced by Mycoplasma gallisepticum infection

doi: 10.1016/j.psj.2025.105485

Figure Lengend Snippet: Influence of AR on NF-κB and NLRP3 proteins in MG induced lung injury. (A) NLRP3 protein in chicken lung tissue. (B) NF-κB protein in chicken lung tissue. (C) Average fluorescence intensity of NLRP3. (D) Localization of NF-κB protein. (E) Levels of NF-κB and NLRP3 proteins in chicken lung tissue, with GAPDH used as a reference. (F) Expression of NLRP3 protein. (G) Ratio of phosphorylated P65 to P65 protein.

Article Snippet: The membranes were incubated overnight at 4°C with NLRP3 polyclonal antibody (1:1000, WL02635, WANLEIBIO, Shenyang, China), NF-κB p65 polyclonal antibody (1:500, WL01273b, WANLEIBIO, Shenyang, China), phosphorylated NF-κB p65 polyclonal antibody (1:600, WL02169, WANLEIBIO, Shenyang, China), Bcl-2 polyclonal antibody (1:900, 12789-1-AP, Proteintech, Chicago, IL), Bax polyclonal antibody (1:900, 50599-2-IG, Proteintech, Chicago, IL), and GAPDH polyclonal antibody (1:5,000, ABclonal, China).

Techniques: Fluorescence, Expressing

Journal: International Journal of Molecular Medicine

Article Title: TNF-α induces premature senescence in tendon stem cells via the NF-κB and p53/p21/cyclin E/CDK2 signaling pathways

doi: 10.3892/ijmm.2025.5581

Figure Lengend Snippet: Effects of the NF-κB and p53/p21/cyclin E/CDK2 signaling pathways on senescence in TNF-α-treated TSCs. (A) ROS staining of TSCs using DCF fluorescence probe, showing intracellular ROS distribution. Scale bar=100 µ m. (B) Quantitative analysis of DCF fluorescence intensity, demonstrating TNF-α-induced elevation of ROS levels. (C) Immunofluorescence staining of γ-H2A.X. Following stimulations with TNF-α (20 ng/ml, six times), the proportion of γ-H2A.X-positive TSCs exhibited a considerable increase. Scale bar=100 µ m. (D) Quantitative analysis of γ-H2A.X-positive TSCs following TNF-α treatment. (E) Expression of γ-H2A.X, H2A.X, p-p65 and p65 following TNF-α stimulation as assessed by western blot. GAPDH was used as a control. (F) Bar groups showed the relative density of γ-H2A.X, H2A.X, p-p65 and p65. (G) Expression of p53, p21, cyclin E and CDK2 following TNF-α stimulation as assessed by western blot. GAPDH was used as a control. (H) Relative density of p53, p21, cyclin E and CDK2. (I) Immunofluorescence examination of p65, p53 and p21 expression was consistent with western blotting. Scale bar=100 µ m. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. ROS, reactive oxygen species; TSCs, tendon stem cells; p-, phosphorylation; ns, not significant.

Article Snippet: After washing by Tris-Buffered Saline with Tween 20 (TBST), membranes were incubated with secondary antibodies (1:10,000) (cat. no. D110087, D110058, Sangon Biotech) at room temperature for 2 h. Primary antibodies included GAPDH (cat. no. 10494-1-AP, anti-rabbit, Proteintech Group, Inc.), p65 (cat. no. sc-8008, anti-mouse, Santa Cruz Biotechnology), p53 (cat. no. ab26, anti-mouse, Abcam), p21 (cat. no. ab188224, anti-rabbit, Abcam), phosphorylated p65 (p-p65; cat. no. sc-136548, anti-mouse, Santa Cruz Biotechnology), γ-H2A.X (cat. no. ab81299, anti-rabbit, Abcam), CDK2 (cat. no. ab32147, anti-rabbit, Abcam) and cyclin E (cat. no. 11554-1-AP, anti-rabbit, Proteintech Group, Inc.), H2A.X (cat. no. 10856-1-AP, anti-rabbit, Proteintech Group, Inc.).

Techniques: Protein-Protein interactions, Staining, Fluorescence, Immunofluorescence, Expressing, Western Blot, Control, Phospho-proteomics

Journal: International Journal of Molecular Medicine

Article Title: TNF-α induces premature senescence in tendon stem cells via the NF-κB and p53/p21/cyclin E/CDK2 signaling pathways

doi: 10.3892/ijmm.2025.5581

Figure Lengend Snippet: Effects of etanercept on the NF-κB and p53/p21/cyclin E/CDK2 signaling pathways in tendon stem cell senescence. (A) ROS staining of TSCs. ROS generation was markedly elevated following repeated TNF-α stimulation and subsequently reduced after repeated administration of etanercept. Scale bar=100 µ m. (B) Quantitative analysis of DCF fluorescence intensity. (C) Expression of γ-H2A.X, H2A.X, p53, p21, p-p65 and p65 after stimulation with TNF-α + etanercept as assessed by western blotting. GAPDH was used as a control. (D) Bar groups showed the relative density of γ-H2A.X, H2A.X, p53, p21, p-p65 and p65. (E) Immunofluorescence of γ-H2A.X and p65 yielded consistent results with those from western blotting. Scale bar=100 µ m. (F) Immunofluorescence of p53 and p21 yielded consistent results with those from western blotting. Scale bar=100 µ m. . ** P<0.01, *** P<0.001, **** P<0.0001. ROS, reactive oxygen species; p-, phosphorylation; ns, not significant.

Article Snippet: After washing by Tris-Buffered Saline with Tween 20 (TBST), membranes were incubated with secondary antibodies (1:10,000) (cat. no. D110087, D110058, Sangon Biotech) at room temperature for 2 h. Primary antibodies included GAPDH (cat. no. 10494-1-AP, anti-rabbit, Proteintech Group, Inc.), p65 (cat. no. sc-8008, anti-mouse, Santa Cruz Biotechnology), p53 (cat. no. ab26, anti-mouse, Abcam), p21 (cat. no. ab188224, anti-rabbit, Abcam), phosphorylated p65 (p-p65; cat. no. sc-136548, anti-mouse, Santa Cruz Biotechnology), γ-H2A.X (cat. no. ab81299, anti-rabbit, Abcam), CDK2 (cat. no. ab32147, anti-rabbit, Abcam) and cyclin E (cat. no. 11554-1-AP, anti-rabbit, Proteintech Group, Inc.), H2A.X (cat. no. 10856-1-AP, anti-rabbit, Proteintech Group, Inc.).

Techniques: Protein-Protein interactions, Staining, Fluorescence, Expressing, Western Blot, Control, Immunofluorescence, Phospho-proteomics

Journal: International Journal of Molecular Medicine

Article Title: TNF-α induces premature senescence in tendon stem cells via the NF-κB and p53/p21/cyclin E/CDK2 signaling pathways

doi: 10.3892/ijmm.2025.5581

Figure Lengend Snippet: Impact of TNF-α on TSCs in normal tendon tissues. Under physiological conditions, TSCs exhibit typical functionality, characterized by regular cell cycles, intact F-actin structures, low levels of ROS and normal expressions of transcription factors. Following stimulation by TNF-α, TSCs experience increased ROS production and DNA damage, activation of the NF-κB signaling pathway (resulting in elevated levels of p-p65 and p65, leading to p65 translocation to the nucleus) and modulation of the p53/p21/cyclin E/CDK2 signaling pathways (resulting in upregulation of p53 and p21 and downregulation of cyclin E and CDK2). These changes induce senescence in TSCs, characterized by alterations such as enlarged cell volume and disrupted F-actin structures. Etanercept, a TNF-α inhibitor, mitigates these effects by binding to TNF-α, thereby inhibiting the activation of signaling pathways. This inhibition leads to reduced ROS levels, mitigates DNA damage, decreases expression of p53, p21 and p-p65, normalizes cyclin E and CDK2 expression and ultimately reverses senescence in TSCs, thereby restoring normal cellular functions. Figure created using BioRender ( app.biorender.com/illustrations ). TSC, tendon stem cells; ROS, reactive oxygen species; F-actin, filamentous-actin; p-, phosphorylation; TNFR, tumor necrosis factor receptor; SA-β-gal, senescence-associated β-galactosidase.

Article Snippet: After washing by Tris-Buffered Saline with Tween 20 (TBST), membranes were incubated with secondary antibodies (1:10,000) (cat. no. D110087, D110058, Sangon Biotech) at room temperature for 2 h. Primary antibodies included GAPDH (cat. no. 10494-1-AP, anti-rabbit, Proteintech Group, Inc.), p65 (cat. no. sc-8008, anti-mouse, Santa Cruz Biotechnology), p53 (cat. no. ab26, anti-mouse, Abcam), p21 (cat. no. ab188224, anti-rabbit, Abcam), phosphorylated p65 (p-p65; cat. no. sc-136548, anti-mouse, Santa Cruz Biotechnology), γ-H2A.X (cat. no. ab81299, anti-rabbit, Abcam), CDK2 (cat. no. ab32147, anti-rabbit, Abcam) and cyclin E (cat. no. 11554-1-AP, anti-rabbit, Proteintech Group, Inc.), H2A.X (cat. no. 10856-1-AP, anti-rabbit, Proteintech Group, Inc.).

Techniques: Activation Assay, Translocation Assay, Protein-Protein interactions, Binding Assay, Inhibition, Expressing, Phospho-proteomics