DFNA5/GSDME Antibody - N-terminal - #AF4016

Fig. 3 Pyroptosis induced by iron accumulation may occur in splenic B cells after SCI. a Three days after SCI, serum samples were collected for quantification of the protein expression levels of MCP-1, IL-1β, IL-6, and TNF-α by ELISA. b Three days after SCI, injured spinal cord homogenates were collected for quantification of the protein expression levels of MCP-1, IL-1β, IL-6, and TNF-α by ELISA. c Three days after SCI, the spleen was stained with Prussian blue to detect the level of iron ion. d Three days after SCI, serum samples were collected to quantify the concentration of iron ions. e Three days after SCI, B cell lysates were collected to quantify the concentration of iron ions. f Detection of Tom20-Bax-caspase-GSDME pathway-related protein expression in B cells by western blot. g The knock-down efficiency of AAV on Tom20 in B cells was verified. h, l, m The expression levels of MCP-1, IL-1 β, IL-6, TNF- α, IgG and IgM in serum of different groups were detected by ELISA. i–k Three days after SCI, the spleens of mice were observed, and the spleen length and organ index of different groups were compared. n, o, q The spleen was taken for HE staining, Prussian blue staining, and immunofluorescence. CD19+ was red, dapi was blue, and merge was combined. p Detection of nerve evoked potentials in different groups of mice. r Detection of the expression of Tom20-Bax-caspsae-GSDME pathway-related proteins in different groups of B cells. All data are expressed as the mean ± SD (n ≥ 3 replicates per group). ns P > 0.05, * P 

Figure 6 - Lip-1 induces pyroptosis in K562 leukemia cells via caspase-3/GSDME activation. K562 cells were treated with 10 and 20 µM Lip-1 for 24 h in most experiments; part F shows an experiment in which K562 cells were pretreated with z-DEVD-FMK for 3 h and further treated with 20 µM Lip-1 for 12 h. (A) K562 cells double-stained with Annexin V-FITC/PI for flow cytometry. (B) Expression levels of TNF-α assessed by real-time polymerase chain reaction and normalized to RPLP0. (C) Protein levels of TNF-α in cell culture supernatants and cell lysates detected by enzyme-linked immunosorbent assay (minimum detectable concentration, 6.9 pg/ml). (D) BAX, cleaved caspase-3, and cleaved PARP protein levels quantified by western blotting. (E) Lip-1 induced caspase-3, but not caspase-1, activation in a concentrationand time-dependent manner. Caspase-1/-3 activities were measured by colorimetric assay. (F) Comparison of cell viability and the protein level of N-terminal GSDME in Lip-1-treated K562 cells and the effects of z-DEVD-FMK inhibition. SH-SY5Y whole cell lysate was used as a positive control. (G) Expression levels of GSDME assessed by real-time polymerase chain reaction and normalized to RPLP0. (H) GSDME and cleaved N-terminal GSDME protein levels quantified by western blotting. SH-SY5Y whole cell lysate was used as a positive control. (I) TUNEL assay used to determine DNA fragmentation. Specimens treated with DNase Ⅰ were used as positive controls. Data represent the mean ± SD from at least three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. Control. Lip-1, liproxstatin-1; ns, not significant.