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: Journal of Animal Science and Biotechnology

Article Title: Proinflammatory polarization of adipose tissue macrophages in cows with subclinical ketosis constitutes a critical driver of adipose tissue remodeling and inflammation

doi: 10.1186/s40104-025-01252-3

Figure Lengend Snippet: Adipose tissue inflammatory response in healthy cows and SCK cows. A The concentrations of (i) tumor necrosis factor (TNF)-α, (ii) lipopolysaccharide binding protein (LBP), (iii) interferon-gamma (IFN-γ) in adipose tissue homogenate of healthy ( n = 6) and SCK group ( n = 6). B Representative Western blots of toll-like receptor 4 (TLR4), phosphorylated nuclear factor kappa B subunit (p-NFκB), NFκB, phosphorylated inhibitor of nuclear factor kappa B kinase subunit beta (p-Iκb), Iκb, TNF-α, NLR Family Pyrin Domain Containing 3 (NLRP3), Caspase1 and β-actin in the adipose tissue of healthy ( n = 6) and SCK ( n = 6) cows. C (i) Quantification of protein levels of TLR4, p-NFκB, NFκB, p-Iκb, Iκb, TNF-α, NLRP3 and Caspase1. The ratios of (ii) p-NFκB/NFκB, (iii) p-Iκb/Iκb. D Representative images (scale bar = 100 μm) of immunofluorescence for TLR4 (green) and DAPI (blue). E Quantification of TLR4 fluorescence intensity (a.u.) in the adipose tissue of healthy ( n = 6) and SCK ( n = 6) cows. F Representative images (scale bar = 50 μm) of immunofluorescence for NFκB (green) and DAPI (blue). G Quantification of NFκB fluorescence intensity (a.u.) in the adipose tissue of healthy ( n = 6) and SCK ( n = 6) cows. Data are presented as mean ± SEM, * P < 0.05, ** P < 0.01; statistical differences were assessed by t -test

Article Snippet: The primary antibodies employed in this study comprised TLR4 (1:1,000, bs-1021R, Bioss, Beijing, China), rabbit anti-NFκB polyclonal antibody (1:100; bs-0465R, Bioss), rabbit anti-CD9 polyclonal antibody (1:100; bs-2489R, Bioss, Beijing, China), rabbit anti-CD68 polyclonal antibody (1:100; bs-1432R, Bioss, Beijing, China), mouse anti-CD68 monoclonal antibody (1:100; NB600-985, NOVUS), rabbit anti-CD86 polyclonal antibody (1:100; bs-1035R, Bioss, Beijing, China), NOS2 (1:1,000; 8985-1-AP, Proteintech, St. Louis, MO, USA), rabbit anti-CD206 recombinant antibody (1:100; 81525-1-RR, Proteintech, Chicago, IL, USA), and rabbit anti-IL-10 polyclonal antibody (1:100; bs-6761R, Bioss, Beijing, China).

Techniques: Binding Assay, Western Blot, Immunofluorescence, Fluorescence