Journal: Clinical and Translational Medicine

Article Title: NLK facilitates Caspase‐8 activation to drive macrophage PANoptosis in sepsis

doi: 10.1002/ctm2.70616

Figure Lengend Snippet: NLK deficiency disrupts PANoptosome assembly and augments RIPK1/3‐ dependent necrosome formation in vivo. (A, B) Representative immunoblots of FADD‐ and RIPK1‐associated complexes, together with corresponding input samples, in macrophages isolated from WT and NKO mice at 8 h post‐CLP. Co‐immunoprecipitates were probed for NLK, Caspase‐8, FADD, RIPK1, p‐RIPK1, RIPK3, and p‐RIPK3. (C–I) Quantification analysis of Caspase‐8, FADD, RIPK1, p‐RIPK1, RIPK3, and p‐RIPK3 in FADD‐associated complexes ( n = 3 independent biological replicates). (J–L) Quantification analysis of p‐RIPK1, RIPK3, and p‐RIPK3 in RIPK1‐associated complexes ( n = 3 independent biological replicates). (M) Representative confocal immunofluorescence images of lung macrophages stained for CD68 (green), Caspase‑8 (red), and ASC (cyan). Merged images indicate ASC–Caspase‐8 colocalisation; boxed regions show magnified views. Scale bars: 50 µm. Statistical significance was determined by using one‑way ANOVA with Bonferroni's post hoc test; * p < .05, ** p < .01 and ns indicates p > .05.

Article Snippet: Antibodies against CD68 (28058‐1‐AP), Caspase‐1 (22915‐1‐AP), p‐RIPK1 (66854‐1‐Ig), FADD ( P14906 ‐1‐AP), HA‐Tag (51064‐2‐AP, 66006‐2‐Ig), Flag‐Tag (66008‐4‐Ig, 20543‐1‐AP), and IgG (B900620) were purchased from Proteintech Group, Inc. (Wuhan, China).

Techniques: In Vivo, Western Blot, Isolation, Immunofluorescence, Staining

Journal: Clinical and Translational Medicine

Article Title: NLK facilitates Caspase‐8 activation to drive macrophage PANoptosis in sepsis

doi: 10.1002/ctm2.70616

Figure Lengend Snippet: NLK deficiency impairs PANoptosome assembly and enhances RIPK1/3‐dependent necrosome formation in macrophages. (A, B) Representative immunoblots of FADD‐ and RIPK1‐associated complexes, together with corresponding input samples, in macrophages isolated from WT and NKO mice at 3 h post‐LPS. Co‐IP were probed for NLK, Caspase‐8, FADD, RIPK1, p‐RIPK1, RIPK3, and p‐RIPK3. (C–I) Quantification analysis of Caspase‐8, FADD, RIPK1, p‐RIPK1, RIPK3, and p‐RIPK3 in FADD‐associated complexes ( n = 3 independent biological replicates). (J–L) Quantification analysis of p‐RIPK1, RIPK3, and p‐RIPK3 in RIPK1‐associated complexes ( n = 3 independent biological replicates). (M) Representative confocal immunofluorescence images showing the co‑localisation of RIPK3 (cyan), ASC (green), and Caspase‑8 (red) in PBS‐ or LPS‐treated BMDMs. Merged images indicate RIPK3–ASC–Caspase‐8 colocalisation; boxed regions show magnified views. Scale bars: 25 µm (merged), 10 µm (zoomed). Statistical differences were analysed by one‑way ANOVA with Bonferroni's post hoc test, * p < .05 and ** p < .01.

Article Snippet: Antibodies against CD68 (28058‐1‐AP), Caspase‐1 (22915‐1‐AP), p‐RIPK1 (66854‐1‐Ig), FADD ( P14906 ‐1‐AP), HA‐Tag (51064‐2‐AP, 66006‐2‐Ig), Flag‐Tag (66008‐4‐Ig, 20543‐1‐AP), and IgG (B900620) were purchased from Proteintech Group, Inc. (Wuhan, China).

Techniques: Western Blot, Isolation, Co-Immunoprecipitation Assay, Immunofluorescence

Journal: Infectious Agents and Cancer

Article Title: Inhibition of death-ligand-induced apoptosis in Epstein-Barr virus-encoded small RNAs-expressing human T-cell line

doi: 10.1186/s13027-026-00736-9

Figure Lengend Snippet: Expression of FLIP and expression and phosphorylation of PKR and FADD in Jurkat transformants. Subconfluent culture of Hela cells was incubated with rhIFNα (1,000 U/mL for 18 hrs) and subsequently with calyculin a (100 nM for 15 min). Cells were harvested, and cell lysates were prepared. A 10-μL (for β-actin) or 20-μL (for other molecules) of aliquot of each sample was applied to each well. After electrophoresis in 12% (β-actin) or 10% (other molecules) polyacrylamide gels, proteins were transferred to a membrane, incubated with the antibodies, and visualized as described in materials and methods. The positions of molecular size markers are indicated (in kDa) on the left in the blot probed with anti-FLIP antibody. Jurkat cells express the long and short form of FLIP (arrow heads). J, Jurkat; J/E6, Jurkat/E6; J/L. Jurkat/L; HIC, Hela treated with IFNα and calyculin A; U, U266

Article Snippet: Antibody solutions used in this study were mouse monoclonal antibodies to β-actin (Sigma-Aldrich), caspase-8, IkBα, phospho-IkBα (Ser32/36), and NF-κB p65 (RelA) (all from Cell Signaling Technology, Beverly, MA), rabbit monoclonal antibodies to phospho-NF-κB p65 (Ser536) and FLIP (all from Cell Signaling), and rabbit polyclonal antibodies to FADD, phospho-FADD (Ser194), PKR (all from Cell Signaling Technology), and phospho-PKR (Thr451; Sigma-Aldrich) diluted with 5% BSA in TBST, and rabbit polyclonal antibody to caspase-3 (Santa Crus Biotech, Santa Crus, CA) diluted with Can Get Signal Solution-1 (Toyobo, Osaka, Japan).

Techniques: Expressing, Phospho-proteomics, Incubation, Electrophoresis, Membrane