Human Ninjurin-1 Antibody (758943)

Detection of Ninjurin-1 by Western Blot

Glycine cytoprotection phenocopies NINJ1 knockout and prevents NINJ1 aggregation in a mouse macrophage cell line.Using a CRISPR-Cas9 system, Ninj1 was knocked out of a RAW mouse macrophage cell line engineered to constitutively express ASC (RAW-ASC) and therefore have the capacity to undergo pyroptosis. (A) Western blot demonstrating NINJ1 knockout compared with the parental line. GAPDH is shown as a loading control. (B) Wildtype and NINJ1 knockout cells were induced to undergo pyroptosis with LPS + nigericin treatment with or without glycine. Supernatant LDH was evaluated as a marker of cytotoxicity by colorimetric assay. Cytotoxicity was decreased in glycine-treated (5 mM) wildtype cells comparably to NINJ1 knockout. Glycine (5 mM) treatment of NINJ1 KO cells provided no additional protection to knockout cells without glycine. * p<0.05 by two-sided ANOVA with Tukey’s multiple comparison correction. (C) LPS-primed wildtype RAW-ASC induced to undergo pyroptosis in the presence of glycine demonstrate similar plasma membrane ballooning to NINJ1 knockout RAW-ASC cells induced to undergo pyroptosis. Membrane ballooning is shown in live cells labeled with the plasma membrane dye FM4-64. Scale bar 20 µm. (D) Native-PAGE analysis of endogenous NINJ1 in RAW-ASC macrophages demonstrates a shift to high molecular weight aggregate upon pyroptosis stimulation, which is abrogated by glycine treatment.Figure 1—figure supplement 2—source data 1.Numerical values for experimental data plotted in Figure 1—figure supplement 2.Numerical values for experimental data plotted in Figure 1—figure supplement 2. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36468682), licensed under a CC-BY license. Not internally tested by R&D Systems.