p nfκb  (Cell Signaling Technology Inc)

Journal: Poultry Science

Article Title: CSF3 enhances the innate immune responses to ALV-J infections via NF-κB and interferon pathways

doi: 10.1016/j.psj.2025.105648

Figure Lengend Snippet: CSF3 enhances the immune response to ALV-J via the NFκB signaling pathway. (A) Western blot analysis of p52, p100, phosphorylated p65 (p-p65), IκBα, and phosphorylated IκBα (p-IκBα) protein following CSF3 overexpression in DF-1 cells. (B) Quantification of protein levels in (A) based on relative grayscale values. (C) Western blot analysis of p52, p100, phosphorylated p65 (p-p65), IκBα, and phosphorylated IκBα (p-IκBα) protein following CSF3 knockdown in DF-1 cells. (D) Quantification of protein levels in (C) based on relative grayscale values. (D, E) RT-qPCR analysis of TNF-α, IL-1β , and IL-6 mRNA expression following CSF3 overexpression (D) or knockdown (E) in DF-1 cells. (F, G) ELISA measurement of TNF-α, IL-1β, and IL-6 protein levels following CSF3 overexpression (F) or knockdown (G) in DF-1 cells. (H, I) RT-qPCR analysis of TNF-α, IL-1β , and IL-6 mRNA expression following CSF3 overexpression (H) or knockdown (I) in CEF cells. (J, K) ELISA measurement of TNF-α, IL-1β, and IL-6 protein levels following CSF3 overexpression (J) or knockdown (K) in CEF cells. (L) Western blot analysis of STAT3, phosphorylated STAT3 (p-STAT3), env, β-actin, IκBα, p-IκBα, p-p65, p52, and p100 after STAT3 phosphorylation inhibition in CSF3-overexpressing DF-1 cells. (M) Quantification of protein levels in (L) based on relative grayscale values. Statistical significance was determined using a two-tailed unpaired Student’s t-test ( p < 0.05). * p < 0.05, **p < 0.01, *** p < 0.001.

Article Snippet: Mouse Anti-ALV-J envelope protein JE9 (kindly provided by Prof. Aijian Qin, Yangzhou University, Yangzhou, China), Rabbit Anti-STAT3 antibody (bs-1141R; Boss, China; 1:1000), Rabbit Anti-phospho-STAT3 (Ser727) antibody (bs-3429R; Boss, China; 1:1000), Rabbit Anti-NFκB2 antibody (10037P, Boss, China; 1:1000), Rabbit Anti-NFκB p-p65 (bs-0982R, Boss, China; 1:1000), Rabbit Anti-IKBα Rabbit (10268-1-AP, Proteintech, USA; 1:1000), Anti-p-IKBα (bs-2513R, Boss, China; 1:1000), and goat anti-rabbit IgG/HRP (bs13278), goat anti-mouse IgG/HRP (bs12478), goat Anti-Mouse IgG ( H + L ) FITC (bs10950) secondary antibody were purchased from Bioss (Beijing, China).

Techniques: Western Blot, Over Expression, Knockdown, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Inhibition, Two Tailed Test

Journal: International journal of molecular sciences

Article Title: NF-κB Activation Is Essential for Cervical Cell Proliferation and Malignant Transformation.

doi: 10.3390/ijms26062493

Figure Lengend Snippet: Figure 1. NF-κB is progressively activated in normal, hyperplastic, CIN1, CIN2, CIN3, and CSCC tissues. (A) Immunohistochemical analysis of total RELA protein expression in normal, hyperplastic, CIN1, CIN2, CIN3, and CSCC tissues. Scale bars: 200 µm for the left panels (100×) and 50 µm for the right panels (400×). (B) Statistical analysis of immunohistochemcal staining intensity (Mean OD) for total RELA in normal, hyperplastic, CIN1-3, and CSCC tissues. (C) Statistical analysis of the percentage of nuclear positivity for total RELA in normal, hyperplastic, CIN1-3, and CSCC tissues. (D) Immunohistochemical analysis of p-RELA protein expression in normal, hyperplastic, CIN1, CIN2, CIN3, and CSCC tissues. Scale bars: 200 µm for the left panels (100×) and 50 µm for the right panels (400×). (E) Statistical analysis of immunohistochemical staining intensity for p-RELA in normal, hyperplastic, CIN1-3, and CSCC tissues. (F) Statistical analysis of the percentage of nuclear positivity for p-RELA in normal, hyperplastic, CIN1-3, and CSCC tissues. ns: no significance, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The cells were then incubated overnight at 4 ◦C with primary antibodies against phospho-RELA (ser536) (sc-136548, Santa Cruz Biotechnolog, Santa Cruz, CA, USA), FN1 (WL00712a, Wanleibio, Shenyang, China), PLAU (WL02483, Wanleibio, China), ICAM1 (WL02268, Wanleibio, China), and Ki67 (WL1384a, Wanleibio, China).

Techniques: Immunohistochemical staining, Expressing, Staining

Journal: International journal of molecular sciences

Article Title: NF-κB Activation Is Essential for Cervical Cell Proliferation and Malignant Transformation.

doi: 10.3390/ijms26062493

Figure Lengend Snippet: Figure 3. Short-term activation of NF-κB promotes the proliferation and migration of HcerEpic cells. (A) RT-qPCR experiment detecting the upregulation of RELA mRNA levels in HcerEpic cells treated with LPS (500 ng/mL). (B) Immunocytochemical experiment detecting the positive expression levels of RELA and p-RELA in HcerEpic cells stimulated by LPS. ImageJ software was used to quantify the percentage area (%Area) of positive expression areas of RELA and p-RELA. (C) CCK-8 assay detecting the effect of different concentrations of LPS on the proliferative ability of HcerEpic cells. (D) Calculation of the EC50 curve for HcerEpic cells treated with LPS based on cell viability. (E) LPS treatment promotes the expression of Ki67 protein in HcerEpic cells. (F) SC75741 treatment inhibits the promoting effect of LPS on the expression of RELA and p-RELA in HcerEpic cells. (G) Detection of HcerEpic cell viability under different concentrations of SC75741 and LPS co-treatment. (H) Scratch wound healing assay detecting the changes in migration ability of HcerEpic cells after LPS and SC75741 treatment. Scale bar: 20 µm. ** p < 0.01, *** p < 0.001.

Article Snippet: The cells were then incubated overnight at 4 ◦C with primary antibodies against phospho-RELA (ser536) (sc-136548, Santa Cruz Biotechnolog, Santa Cruz, CA, USA), FN1 (WL00712a, Wanleibio, Shenyang, China), PLAU (WL02483, Wanleibio, China), ICAM1 (WL02268, Wanleibio, China), and Ki67 (WL1384a, Wanleibio, China).

Techniques: Activation Assay, Migration, Quantitative RT-PCR, Expressing, Software, CCK-8 Assay, Wound Healing Assay

Journal: International journal of molecular sciences

Article Title: NF-κB Activation Is Essential for Cervical Cell Proliferation and Malignant Transformation.

doi: 10.3390/ijms26062493

Figure Lengend Snippet: Figure 4. Long-term activation of NF-κB promotes the expression of oncogenes in HcerEpic cells. (A) Immunocytochemical experiment detecting the changes in protein expression of p-RELA, Ki67, FN1, PLAU, and ICAM1 in HcerEpic cells after 1, 3, and 7 days of LPS treatment. (B) Western blot experiment detecting the changes in protein expression of RELA, p-RELA, FN1, PLAU, and ICAM1 in HcerEpic cells after 1, 3, and 7 days of LPS treatment. (C) Immunocytochemical experiment detecting the changes in protein expression of p-RELA, Ki67, FN1, PLAU, and ICAM1 in HcerEpic cells treated with LPS alone and in combination with SC75741. Scale bar: 20 µm. ns: no significance, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The cells were then incubated overnight at 4 ◦C with primary antibodies against phospho-RELA (ser536) (sc-136548, Santa Cruz Biotechnolog, Santa Cruz, CA, USA), FN1 (WL00712a, Wanleibio, Shenyang, China), PLAU (WL02483, Wanleibio, China), ICAM1 (WL02268, Wanleibio, China), and Ki67 (WL1384a, Wanleibio, China).

Techniques: Activation Assay, Expressing, Western Blot

Journal: International journal of molecular sciences

Article Title: NF-κB Activation Is Essential for Cervical Cell Proliferation and Malignant Transformation.

doi: 10.3390/ijms26062493

Figure Lengend Snippet: Figure 6. NF-κB regulates the expression of oncogenic DEGs and malignant phenotypes in cervical squamous cancer cells. (A,B) Western blot detecting the expression of RELA, p-RELA, E-cadherin, N- cadherin, FN1, ICAM1, and PLAU in SiHa and CasKi cells. (C) CCK-8 assay detecting the proliferative ability of SiHa and CasKi cells after treatment with different concentrations of SC75741 for 24 and 48 h. (D) Representative images of wound healing in SiHa and CasKi cells after scratch injury for 36 and 72 h in control and SC75741 treatment groups. Scale bar: 100 µm. (E) Colonies formed by SiHa and CasKi cells in control and SC75741 treatment groups. Data represent three replicates of a representative experiment. ns: no significance, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The cells were then incubated overnight at 4 ◦C with primary antibodies against phospho-RELA (ser536) (sc-136548, Santa Cruz Biotechnolog, Santa Cruz, CA, USA), FN1 (WL00712a, Wanleibio, Shenyang, China), PLAU (WL02483, Wanleibio, China), ICAM1 (WL02268, Wanleibio, China), and Ki67 (WL1384a, Wanleibio, China).

Techniques: Expressing, Western Blot, CCK-8 Assay, Control