Journal: International Journal of Biological Sciences
Article Title: Ganglioside GA2-mediated caspase-11 activation drives macrophage pyroptosis aggravating intimal hyperplasia after arterial injury
doi: 10.7150/ijbs.97106
Figure Lengend Snippet: GA2 mediates Cytochrome C-Casp9-Casp3 pathway activation through Cl-Casp4/11 binding to and cleaving BID. (A) Representative immunoblots of Casp8, Cl-Casp8, Casp9, Cl-Casp9, Casp3 and Cl-Casp3 in PMs and analysis results (n = 5). (B) Representative immunoblots of cytochrome C (Cyt C) in PM cytosol (Cyto) and mitochondrion (Mito) and analysis results (n = 5). (C) Representative immunoblots of BID, tBID (truncated BID), BIM and PUMA in PMs and analysis results (n = 5). (D-E) Coimmunoprecipitation (Co-IP) of activated Casp4 binds to BID in HEK293T cells (n = 3). HEK293T cells were transfected with the indicated plasmids for 24 h and the lysates incubated with anti-Flag-magnetic-beads, then immunoblotted with antibodies against Flag or HA. (F) Representative immunoblots of BID, tBID, Casp3, and Cl-Casp3 in PMs and analysis results (n = 6). (G) Representative immunoblots of BID, tBID, Casp3, and Cl-Casp3 in RAW264.7 cells and analysis results (n = 5). (H) Representative immunofluorescence staining sections from mice after 7 d of the injury for BID (light gray), F4/80 (green), and Casp11 (red) (n = 7). Nuclei were stained with DAPI (blue). Scale bars: 100 μm. A , B , C , F (tBID) and G (tBID) were tested using a One-way ANOVA test; F (Cl-Casp3) and G (Cl-Casp3) were tested using a Welch's ANOVA test.
Article Snippet: The following primary antibodies were used for western blot: MLKL (1:1000, 37333S, CST), Phospho-MLKL (1:1000, 37333S, CST), RIPK3 (1:1000, 95702S, CST), Phospho-RIPK3 (1:1000, 91702S, CST), GSDMD (1:500, sc-393581, Santa Cruz), GSDME (1:1000, ab215191, Abcam), caspase-11 (1:1000, ab180673, Abcam), caspase-1 (1:1000, ab179515, Abcam), caspase-4 (1:1000, M029-3, MBL), caspase-8 (1:1000, 9746T, CST), caspase-9 (1:1000, 9508T, CST), caspase-3 (1:1000, 9662S, CST), cytochrome C (1:1000, 11940S, CST), BID (1:1000, AF860, R&D Systems), BIM (1:500, sc-374358, Santa Cruz), PUMA (1:500, sc-374223, Santa Cruz), COX IV (1:5000, 11242-1-AP, Proteintech), β-actin (1:5000, BS6007M, Bioworld).
Techniques: Activation Assay, Binding Assay, Western Blot, Co-Immunoprecipitation Assay, Transfection, Incubation, Magnetic Beads, Immunofluorescence, Staining