Journal: Journal of dairy science

Article Title: Sodium butyrate reduces bovine mammary epithelial cell inflammatory responses induced by exogenous lipopolysaccharide, by inactivating NF-κB signaling.

doi: 10.3168/jds.2020-18189

Figure Lengend Snippet: Figure 5. Effects of sodium butyrate (SB) on NF-κB signaling pathway. Bovine mammary epithelial cells (MAC-T) were treated with 1 µg/mL LPS for 6 h; with 0.5 mM SB for 18 h; or with 0.5 mM SB for 18 h, followed by a challenge with 1 µg/mL LPS for an additional 6 h. (A) Western blot analysis of p-IκB α, IκB α, p-p65, and p65. (B) Relative protein expression levels of p-IκB α to IκB α. (C) Relative protein expression levels of p-p65 to p65. Data from the control group were used to normalize the data of each treatment group. Comparisons among groups were calculated using a one-way ANOVA with subsequent Bonferroni correction. Data presented are mean ± SEM. Different lowercase letters in bar charts indicate significant differences (P < 0.05).

Article Snippet: Membranes were then incubated with antibodies against phosphorylatd-IκB α (p-IκB α; sc-271980, Santa Cruz Biotechnology; 1:5,000), IκB α (sc-166588, Santa Cruz Biotechnology; 1:1,000), NFκB p65 (sc-8008, Santa Cruz Biotechnology; 1:1,000), phosphorylatd-NF-κB p65 (p-NF-κB p65; sc-166748, Santa Cruz Biotechnology; 1:1,000), acetyl-H3 (8173, Cell Signaling Technology, Danvers, MA; 1:1,000), histone H3 (4499, Cell Signaling Technology; 1:1,000), and GAPDH (ab8245, Abcam, Cambridge, MA; 1:5,000) overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibody (Boster, Wuhan, China) for 45 min at room temperature.

Techniques: Western Blot, Expressing, Control

Journal: Biochimica et biophysica acta

Article Title: Insulin potentiates cytokine-induced VCAM-1 expression in human endothelial cells.

doi: 10.1016/j.bbadis.2008.05.006

Figure Lengend Snippet: Fig. 4. Effects of the proteasome inhibitor lactacystin on VCAM-1 surface expression induced by TNF-α in the presence or absence of insulin in endothelial cells, and effects of TNF-α, with or without insulin, on the phosphorylated and total cellular levels of IκB-α. Panels A and B: VCAM-1 surface expression, at EIA, in cells pretreated with lactacystin (10 μmol/L for 30 min), and then co-incubated with insulin (10−9 to 10−7 mol/L) and TNF-α for 16 h. ⁎pb0.05 vs non-insulin-stimulated control; #pb0.001 vs non-lactacystin treated cells. Results are expressed as mU of optical density, and each concentration point is the mean±S.D. from 3 experiments, each consisting of 8 replicates per condition. B: Immuno- blottings with antibodies against Ser32-phosphorylated (upper panels) or non-phosphory- lated IκB-α (bottom panels) in total proteins extracted from HUVEC incubated with insulin (10−9–10−7 mol/L) for 16 h, with (B) subsequent incubation with 0.1 ng/mL TNF-α for 15 min.

Article Snippet: After blocking, the membranes were incubated overnight at 4 °C with primary antibodies against VCAM-1 (Santa Cruz Biotechnologies, Santa Cruz, CA), and with primary antibodies against the constitutive and Ser32-phosphorylated inhibitor IκB-α (Santa Cruz Biotechnology).

Techniques: Expressing, Incubation, Control, Concentration Assay

Journal: Nature Communications

Article Title: Inflammation-specific DNA origami nanodevice for delivery of siRNAs to treat ulcerative colitis

doi: 10.1038/s41467-025-67183-9

Figure Lengend Snippet: a The fluorescence images of siTNF&siInte (200 nM) and the nanodevice (10 nM) after co-incubation with Caco-2 for 6 h. Scale bars: 10 μm. The siTNF-α was modified with Cy3 (in green), and the siInte α4 was modified with Cyanine 7 (Cy7, in magenta). b RT-qPCR analysis of TNF-α and integrin α4 mRNA levels. RAW264.7 cells treated with or without 500 ng/mL LPS for 12 h, and then co-incubated with 10 nM nanodevice for 6 h. n = 3 biological replicates, **p = 0.0014 TNF-α, **p = 0.0027, integrin α4. c WB analysis of TNF-α and integrin α4 protein levels. RAW264.7 cells were treated with nanotube (10 nM), free siRNA (200 nM) and nanodevice (10 nM) for 36 h. siTNF&siInte represented the mixture of the free siTNF-α and siInte α4, and was directly incubated with cells without liposome-mediated transfection. n = 3 biological replicates, ***/ ### p < 0.0001. d Schematic representation of TNF-α activating NF-κB to promote inflammatory factors expression. e WB analysis of p65/p-p65 and IκB-α/p-IκB-α protein levels. RAW264.7 cells were incubated with 500 ng/mL LPS and then treated with PBS and nanodevice (10 nM) for 24 h. n = 3 biological replicates, ns p = 0.1937, p-p65, **p = 0.0067, p-IκB-α, *p = 0.0104, IκB-α. f The IL-1 β and IL-6 protein levels determined by ELISA kit. RAW264.7 cells were treated with PBS or nanodevice (10 nM) and LPS (500 ng/mL) for 24 h, then cell supernatants were collected for analysis. n = 3 biological replicates, **p = 0.0017, IL-1β, ***p = 0.0003, IL-6. Data are presented as the mean ± SD. Statistical analysis were performed with unpaired two-tailed Student’s t test ( b ) or one way ANOVA with Tukey’s test ( c, e, f ). *p vs PBS. # p vs siTNF&siInte. Source data are provided as a Source Data file.

Article Snippet: The membrane was incubated with primary antibody TNF-α, integrin α4, ZO-1 (Proteintech, #21773-1-AP, China), Occludin (Abclonal, #A24601, China), Iκb-α/p-Iκb-α (Proteintech, #10268-1-AP, #82349-1-RR, China), NF-κB-p65(Proteintech, #10745-1-AP, China) NF-κB-p-p65(Immunoway, #YP0191, USA), cleaved-caspase3 (CST, #9664, USA), PARP (CST, #9532, USA), β- actin (Zen BioScience, #250132, China) overnight at 4 °C, and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 hour.

Techniques: Fluorescence, Incubation, Modification, Quantitative RT-PCR, Transfection, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test