cleaved casp1 mouse  (Cell Signaling Technology Inc)

Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: Saikosaponin A targets HDAC6 to inhibit Mycobacterium tuberculosis-induced macrophage Pyroptosis via autophagy-mediated NLRP3 inflammasome inactivation.

doi: 10.1016/j.phymed.2025.156693

Figure Lengend Snippet: Fig. 3. SSA Suppresses Mtb-Induced Pyroptosis. *p < 0.05, **p < 0.01, ***p < 0.001. NS, nonsignificant. (A, B, C, D) Changes in NLRP3 protein levels were evaluated with Western blotting following Mtb infection in macrophages. (E, F) Protein samples from various treatment groups were cross-linked with DSS and analyzed with Western blotting, revealing monomeric, dimeric, and oligomeric forms of ASC (n = 3). (I, J) Co-immunoprecipitation of ASC followed by immunoblotting for NLRP3 and ASC, with subsequent reblotting for NLRP3 and ASC. One representative experiment of three is shown. (K, l) Changes in CASP1 (p20) and GSDMD-N protein levels were evaluated with Western blotting after cells were treated with 8 μM SSA for 6, 12, or 24 h following 4 h of Mtb infection. (G, H) IL-1β levels were measured using ELISA (n = 3) after cells were treated with 2-8 μM SSA for 12 h following 4 h of Mtb infection.

Article Snippet: For antibodies, we applied NLRP3 (Cat# 15,101, CST), NLRP3 (Cat# A21906, Abclonal), GSDMD (Cat# 39,754, CST), cleaved CASP1 (mouse) (Cat# 89,332, CST), cleaved CASP1 (human) (Cat# AG-20B0048-C100, AdipoGen), HDAC6 (Cat# 7612, CST), LC3B (Cat# 43,566, CST), LC3B Mouse mAb (Cat# A17424, Abclonal), LC3B (Cat# SC-271,625 Santa Cruz), p62(Cat# A11250, Abclonal), mTOR (Cat# 2972, CST), p-mTOR (Cat# 5536, CST), p-ULK1 Ser757 (Cat# 14,202, CST), p-ULK1 Ser555 (Cat# 5869, CST), ULK1 (Cat# 8054, CST), AMPKα (Cat# 5831, CST), p-AMPKα Thr172 (Cat# 2535, CST), Alexa Fluor 594-conjugated goat anti-mouse IgG (Cat# 8890S, CST), Alexa Fluor 488 goat anti-rabbit IgG (Cat# AS053, Abclonal), β-actin (Cat# CL594–66,009, ProteinTech), ASC (Cat# SC-514,414, Santa Cruz), ASC (Cat# A16672, Abclonal), HRP-conjugated AffiniPure Mouse AntiRabbit IgG Light Chain (Cat# AS061, Abclonal), alpha-tubulin (acetyl K40) (Cat# ab179484, Abcam), Acetyl-α-tubulin (Lys K40) (Cat# 5335, CST), and DYKDDDDK Tag (D6W5B) Rabbit mAb (Cat# 14,793, CST).

Techniques: Western Blot, Infection, Immunoprecipitation, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: Artificial cells delivering itaconic acid induce anti-inflammatory memory-like macrophages to reverse acute liver failure and prevent reinjury

doi: 10.1016/j.xcrm.2023.101132

Figure Lengend Snippet: AI-Cells controlled liver injury by inhibiting macrophage NLRP3 inflammasome activation and reducing IL-1β production (A) The levels of IL-1β, TNF-α, and IL-6 in the supernatant of BMDMs/Kupffer cells in various groups (n = 3 biological replicates). (B) Schematic representation of a BMDM/Kupffer cell inflammation model responding to TNF-α, IL-6, and IL-1β. AI-Cell treatment significantly reduced the concentration of IL-1β but had no significant effect on TNF-α and IL-6. (C) Quantification of viable AML-12 cells after IL-1β exposure (n = 5 biological replicates). The AML-12 cells were cultured in the B-AI-Cells CM supplemented with recombinant IL-1β. B-LPS&ATP CM was set up as a control. (D) Survival of AML-12 cells after K-LPS&ATP CM and IL-1β antibody exposure (n = 3 biological replicates). K-LPS&ATP CM was set up as a control. (E) Representative microscopy images of AML-12 cells treated with various conditioned medium from Kupffer cells and stained with crystal violet. Scale bars: 1,000 μm. (F) Western blot of NLRP3, pro-Casp1, Casp1 p20, and β-actin with protein lysates from liver tissues of sham, APAP, and AI cells groups. (G) Western blot of IL-1β with protein lysates from liver tissues of sham, APAP, and AI-Cells groups. (H–K) Densitometric analysis of NLRP3, pro-Casp1, Casp1 p20, and IL-1β proteins by ImageJ software (n = 3 biological replicates). (L) IL-1β staining of liver tissues from the sham, APAP, or AI-Cell-treated ALF mice after euthanasia. Scale bars: 200 μm. (M) Schematic diagram of AI-Cells blocking activation of NLPR3 inflammasome. (N) Docking mode of NLRP3 (white surface and green cartoon) with itaconic acid (cyan sticks). (O) Three-dimensional (3D) interaction mode of NLRP3 (green cartoon) and itaconic acid (cyan sticks). The key residues are shown as green sticks and the hydrogen bond is shown in yellow dashed lines. All data are expressed as mean ± SD; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, and ns is p > 0.05 by one-way ANOVA.

Article Snippet: Anti-pro-Casp1/anti-Casp1 p20 antibody (mouse) , Cell Signaling Technology , Cat# 89332; RRID: AB_2923067.

Techniques: Activation Assay, Concentration Assay, Cell Culture, Recombinant, Control, Microscopy, Staining, Western Blot, Software, Blocking Assay

Journal: Cell Reports Medicine

Article Title: Artificial cells delivering itaconic acid induce anti-inflammatory memory-like macrophages to reverse acute liver failure and prevent reinjury

doi: 10.1016/j.xcrm.2023.101132

Figure Lengend Snippet:

Article Snippet: Anti-pro-Casp1/anti-Casp1 p20 antibody (mouse) , Cell Signaling Technology , Cat# 89332; RRID: AB_2923067.

Techniques: Recombinant, RNA Extraction, Staining, Real-time Polymerase Chain Reaction, Software