anti nfat4  (R&D Systems)

Journal: Journal of medicinal chemistry

Article Title: A Peptidyl Inhibitor that Blocks Calcineurin-NFAT Interaction and Prevents Acute Lung Injury

doi: 10.1021/acs.jmedchem.0c01236

Figure Lengend Snippet: Effect of CNI93 on NFATc3 nuclear translocation and cytokine secretion ex vivo. (A,B) Mouse bone marrow-derived macrophages were pretreated with 1 μM biotinylated CPP9-VAVAA (A) or CNI93 (B) and stimulated with LPS (100 ng/mL) or PBS for 1 h. Intracellular NFATc3 (green) and biotinylated peptide (red) were visualized using Nikon A1R microscope equipped with 488 nm and 543 nm excitation lasers. Single confocal optical sections (pinhole set to achieve 1 Airy unit) are shown (n = 7–10 cells/group). Scale bars, 10 μm. (C,D) Inhibition of LPS-induced TNFα and IL-6 secretion by CNI93. Total lung macrophages from healthy mice were pretreated for 4 h with indicated concentrations of DMSO, CPP9-VAVAA, CsA, or CNI93 and stimulated with LPS (100 ng/mL for 8 h) or PBS. Statistical significance determined by one-way ANOVA to vehicle (0.1% DMSO) treated cells, *, p < 0.05, **, p < 0.01, ***, p < 0.001.

Article Snippet: Antibodies for immunofluorescence and flow cytometry were purchased from the following suppliers: α-mouse CD45-PerCP/Cy5.5 and streptavidin-Alexa Fluor 594 from Biolegend (Cat. No. NC0217834 and 405240), α-rabbit IgG-Alexa Fluor 488 from Cell Signaling (Cat. No. 4412S), and Human/Mouse/Rat NFATc3 from R&D Systems (Cat. No. AF5834).

Techniques: Translocation Assay, Ex Vivo, Derivative Assay, Microscopy, Inhibition