Journal: International Journal of Molecular Sciences

Article Title: In Vitro and In Vivo Validation of Endothelium-Derived Potential Therapeutics for Myocardial Ischemia/Reperfusion Injury Identified by an AI-Enhanced Single-Cell and Virtual-Cell Paradigm

doi: 10.3390/ijms27062743

Figure Lengend Snippet: Andrographolide inhibits the S100A8 downstream IL-17 signaling pathway and reduces the inflammatory response of H/R-induced CMECs. ( A , B ) The expressions of IL-17A, IL-17RA, Act1, and NF-κB proteins were determined by Western blot ( n = 3). ( C – E ) The levels of IL-1β, IL-6, and TNF-α were determined through ELISA. ( F – H ) The expressions of IL-17A, IL-17RA and Act1 proteins were determined by Western blot in co-transfection with the overexpression S100A8 plasmid ( n = 3). Data are presented as mean ± SD ( n = 6). “Con or C” stands for the control group, “H/R” or “H” indicates the H/R group, and “H + AG or H + A” means the H/R group treated with AG. “H + A + S” means the H/R group treated with AG and overexpression of S100A8. ** p < 0.01 compared to the Con group; # p < 0.05, ## p < 0.01 compared to the H/R or H group, @ p < 0.05, @@ p < 0.01 compared to the H + A + S group.

Article Snippet: The membranes were blocked for 1 h at room temperature with 5% skimmed milk before being incubated overnight at 4 °C with the following primary antibodies: S100A8 (A15315, Abclonal, Wuhan, China), IL-17A (A12454, Abclonal, Wuhan, China), IL-17RA (A10052, Abclonal, Wuhan, China), Act1 (A6776, Abclonal, Wuhan, China), p-NF-κB p65 (1:500, AP0124, Abclonal, Wuhan, China), NF-κB p65 (1:500, A00284-1, Boster, Wuhan, China), β-actin (1:2000, TA-09, Zhongshan Golden Bridge, Beijing, China), and GAPDH (1:2000, TA-08, Zhongshan Golden Bridge, Beijing, China).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Cotransfection, Over Expression, Plasmid Preparation, Control

Journal: Biology of reproduction

Article Title: Epidermal growth factor-induced proliferation of chicken primordial germ cells: involvement of calcium/protein kinase C and NFKB1.

doi: 10.1095/biolreprod.108.072728

Figure Lengend Snippet: FIG. 6. Effect of EGF on NFKB1 (p65) activation. A) Time dependence of EGF-induced NFKB1 (p65) translocation; PGCs were incubated with 10 ng/ml EGF for 0–120 min. B) PGCs were pretreated with AG1478 (106

Article Snippet: Antibodies against EGF, EGFR, pan-PKC, NFKB1, NFKBIA, BAX, BCL2, caspase 3, caspase 9, biotin-conjugated goat antimouse IgG or anti-rabbit IgG, and streptavidin-biotin-peroxidase complex (SABC) immunoreaction kit were from Boster Inc. (Wuhan, China).

Techniques: Activation Assay, Translocation Assay, Incubation

Journal: Nature Communications

Article Title: Induction of immunosuppressive functions and NF-κB by FLIP in monocytes

doi: 10.1038/s41467-018-07654-4

Figure Lengend Snippet: FLIP activates the canonical NF-κB pathway in myeloid cells. a Suppressive activity of THP1 cells transfected with c-FLIP RNA was measured by enumeration of an absolute number of human CD3 + T cells collected after in vitro co-culture. b Immunofluorescence confocal microscopy of p65 (red), p50 and p52 (green) nuclear translocation in transfected THP1 cells. c Nuclear c-FLIP protein expression by western blot in THP1 cells transfected with either GFP or c-FLIP RNA at different time points. d Immunofluorescence confocal microscopy of p50 (red) and c-FLIP (green) nuclear translocation in transfected THP1 cells. e CD11b + Ly6C + cells (M-MDSCs) were isolated from the spleen of MCA203 tumor-bearing, wild-type mice by flow sorter and transfected for 18 h with scramble, IKKα, IKKβ, or the combination IKKα plus IKKβ siRNAs. After transfection, cells were washed three times. M-MDSCs were co-incubated with peptide-stimulated CellTrace-labeled OT-I cells. Suppressive activity was measured by enumerating absolute numbers of CD8 + T cells collected after in vitro co-culture. f PD-L1 expression in transfected M-MDSCs. Data are presented either as mean ± s.e.m of three independent experiments ( a ) or as mean ± s.e.m of four independent experimental transfections ( e , f ) where each plot refers to CD11b + Ly6C + cells isolated from the pooled spleens of three tumor-bearing mice. Original images × 800 for all panels ( b , d ). * P < 0.05, ** P < 0.01; *** P < 0.001; n.s., not significant, by Mann–Whitney test ( a , b , e , f )

Article Snippet: For immunofluorescence, cyto-spin slides (ShandonTM Single CytoslidesTM, Thermo Fisher Scientific, Waltham, MA, USA) were incubated with the following primary antibodies: NF-kB p65 Mouse mAb (#6956, Cell Signaling, Danvers, MA, USA), NF-kB2 p100/p52 Rabbit mAb (#3017, Cell Signaling, Danvers, MA, USA), polyclonal rabbit anti-NF-κB p105/P50 picoband antibody (PB9149, Boster Biological Technology, CA, USA), anti-NFkB p105/p50 antibody (ab32360, Abcam), and monoclonal mouse anti-FLIP antibody (MAB8430, R&D System).

Techniques: Activity Assay, Transfection, In Vitro, Co-Culture Assay, Immunofluorescence, Confocal Microscopy, Translocation Assay, Expressing, Western Blot, Isolation, Incubation, Labeling, MANN-WHITNEY

Journal: Acta biochimica et biophysica Sinica

Article Title: Therapeutic effect of artemisinin on lupus nephritis mice and its mechanisms.

doi: 10.1093/abbs/gmq101

Figure Lengend Snippet: Figure 2 Expression of NF-kBp65 protein, NF-kB, and TGF-b1 mRNA in renal tissue (A) Activity of NF-kB. (B) NF-kBp65 protein expression. (C) TGF-b1 mRNA expression. In the three figures, (1) stands for the mice in group (i), (2) for the mice in group (ii), (3) for the mice in group (iii), and (4) for the mice in group (iv).

Article Snippet: The membrane was incubated with mouse anti-NF-kBp65 monoclonal antibody (Wuhan Boster Biological Technology, Ltd., Wuhan, China) (1: 200) and then with HRP-conjugated anti-mouse IgG secondary antibody (Wuhan Boster Biological Technology, Ltd.) (1: 1000).

Techniques: Expressing, Activity Assay

Journal: Scientific Reports

Article Title: Promotion of the inflammatory response in mid colon of complement component 3 knockout mice

doi: 10.1038/s41598-022-05708-8

Figure Lengend Snippet: Expression levels of members in the NF-κB signaling pathway. ( A ) Expression levels of NF-κB-p65 and IκB-α proteins were determined by Western blot analysis using specific primary antibody and HRP-labeled anti-rabbit IgG antibody. ( B ) Band intensities were determined using an imaging densitometer, and protein expressions were calculated relative to the intensity of β-actin. Tissue samples were collected from 3 to 5 mice per group, and each lysate was analyzed in duplicate for Western blot (final n = 6–10). Data are reported as the mean ± SD. * indicates p < 0.05 compared to the WT mice.

Article Snippet: Proteins (30 μg) were then separated by 4–20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) for 2 h, and subsequently transferred to nitrocellulose membranes for 3 h at 40 V. Each membrane was then incubated separately, overnight at 4 °C, with the following primary antibodies: anti-C3 (ab200999, Abcam Com.), anti-C3aR (bs-2955R, Bioss Inc.), anti-CR1 (LS-C777464, LSBio Inc.), anti-COX-2 (12282, Cell Signaling Technology Inc., Danvers, MA, USA), anti-NLRP3 (15101, Cell Signaling Technology Inc.), anti-Cas 1 (24232, Cell Signaling Technology Inc.), anti-ASC (67824, Cell Signaling Technology Inc.), anti-iNOS (PA3-0304, Thermo Fisher Scientific Inc.), anti-ERK1/2 (9102, Cell Signaling Technology Inc.), anti-p-ERK (E-4) (9101, Santa Cruz Biotechnology Inc., Dallas, TX, USA), anti-JNK (9252, Cell Signaling Technology Inc.), anti-p-JNK (9251, Cell Signaling Technology Inc.), anti-p38 (9212, Cell Signaling Technology Inc.), anti-p-p38 (9211, Cell Signaling Technology Inc.), anti-p-NF-κB-p65 (A00284T254, Boster Bio Inc., Pleasanton, CA, USA), anti-IκB (9242, Cell Signaling Technology Inc.), anti-p-IκB (9246S, Cell Signaling Technology Inc.), anti-IL-6 (SC-1265, Santa Cruz Biotechnology Inc.), anti-C5 (ab11898, Abcam Com.), anti-thrombin (ab92621, Abcam Com.), anti-E-cadherin (24E10, Cell Signaling Technology Inc.) or anti-β-actin (4967, Sigma-Aldrich Co.).

Techniques: Expressing, Western Blot, Labeling, Imaging