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Santa Cruz Biotechnology fadd
Fadd, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 282 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Article Title: Voluntary exercise training attenuated the middle-aged maturity-induced cardiac apoptosis.
Article Snippet: Aims: Voluntary exercise training has cardioprotective effects in humans, but the underlying mechanism is unknown.. This research was done to estimate the effect of voluntary exercise training to attenuate middle-aged maturity-induced cardiac apoptosis.. Materials and methods: The study was designed to divide 64 male mice randomly into four groups, consisting of a 9-month sedentary pre-middle-aged group (9M), 15-month sedentary middle-aged group (15M), and two exercise groups using a voluntary wheel running respectively (9M+EX, 15M+EX).

Article Title: Cooperation of TRADD- and RIPK1-dependent cell death pathways in maintaining intestinal homeostasis.
Article Snippet: The immunocomplex was captured by 20μl of protein A/G agarose (Invitrogen) 4 h at 4 °C.

Article Title: Angiotensin II Receptor Blocker Irbesartan Enhanced SIRT1 longevity Signaling Replaces the Mitochondrial Biogenetic Survival Pathway to Attenuate Hypertension-Induced Heart Apoptosis
Article Snippet: The membranes were incubated overnight at 4 °C with primary antibodies, including Fas Ligand, Fas, FADD, Bax, cytosolic cytochrome c, active caspase-8, active caspase-9, and active caspase-3, IGF-II, p -JNK, SIRT1, PGC-1, Bcl-2, Bcl-xL, and α-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Article Title: Decreased linear ubiquitination of NEMO and FADD on apoptosis with caspase-mediated cleavage of HOIP.
Article Snippet: Accepted Manuscript Decreased linear ubiquitination of NEMO and FADD on apoptosis with caspasemediated cleavage of HOIP Eiji Goto, Fuminori Tokunaga PII: S0006-291X(17)30308-X DOI: 10.1016/j.bbrc.2017.02.040 Reference: YBBRC 37288 To appear in: Biochemical and Biophysical Research Communications Received Date: 3 February 2017 Accepted Date: 7 February 2017 Please cite this article as: E. Goto, F. Tokunaga, Decreased linear ubiquitination of NEMO and FADD on apoptosis with caspase-mediated cleavage of HOIP, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.02.040.. This is a PDF file of an unedited manuscript that has been accepted for publication.. As a service to our customers we are providing this early version of the manuscript.

Incubation:

Article Title: Cooperation of TRADD- and RIPK1-dependent cell death pathways in maintaining intestinal homeostasis
Article Snippet: .. The lysates were incubated with FADD antibody (Santa Cruz Biotechnology, sc-6036, 1:100) in rotation overnight at 4 °C. .. The immunocomplex was captured by 20 μl of protein A/G agarose (Invitrogen) 4 h at 4 °C.

Article Title: Cooperation of TRADD- and RIPK1-dependent cell death pathways in maintaining intestinal homeostasis.
Article Snippet: .. The lysates were incubated with FADD antibody (Santa Cruz Biotechnology, sc-6036, 1:100) in rotation overnight at 4 °C. .. The immunocomplex was captured by 20μl of protein A/G agarose (Invitrogen) 4 h at 4 °C.

Article Title: The Autophagy-Initiating Kinase ULK1 Controls RIPK1-Mediated Cell Death.
Article Snippet: Cells were lysed in 0.5%Nonidet P-40/IGEPAL CA-630 IP lysis buffer (20 mM Tris-HCl, [pH 7.4], 150 mM NaCl, 0.5% Nonidet P-40/IGEPAL CA-630, 10% glycerol, 1 3 complete protease inhibitor Cocktail (Sigma-Aldrich; P2714), 1 3 PhosSTOP (Roche; 04906837001) and 10 mM PR619) and rotated for 20 minutes at 4 C. After centrifugation at 17,000 g for 10 min, the supernatant was precleared using Protein G Sepharose beads for 30 min at 4 C. After that, the precleared supernatant was incubated with FLAG beads (Sigma-Aldrich; A2220) overnight. .. Beads were washed 4 times using IP buffer and eluted with 23 SDS-Loading buffer for 10 minutes at 95 C. For complex II IP, MEFs and L929 cells were stimulated as indicated in the figure legends and cells were lysed in 1% Triton X-100 IP lysis buffer (30 mM Tris-HCl [pH 7.4], 120mM NaCl, 2 mMEDTA, 2mMKCl, 1%Triton X-100, complete protease-inhibitor cocktail) on ice for 20min and centrifuged at 17,000 g for 10 min. Supernatant was precleared using Protein G Sepharose beads for 30 min at 4 C. 25 mL protein G beads were blocked for 1 h with lysis buffer containing 1%BSA and boundwith 1.5 mg FADD antibody (Santa Cruz Biotechnology; sc-6036) for 2 h and incubated with lysates for 4 h at 4 C or lysates were incubated with 3 mg FADD antibody (Merck Millipore; 05-486) for 6-8 h and then incubated with 25 mL protein G beads blocked for 1 h with lysis buffer containing 1%BSA overnight. ..

Lysis:

Article Title: The Autophagy-Initiating Kinase ULK1 Controls RIPK1-Mediated Cell Death.
Article Snippet: Cells were lysed in 0.5%Nonidet P-40/IGEPAL CA-630 IP lysis buffer (20 mM Tris-HCl, [pH 7.4], 150 mM NaCl, 0.5% Nonidet P-40/IGEPAL CA-630, 10% glycerol, 1 3 complete protease inhibitor Cocktail (Sigma-Aldrich; P2714), 1 3 PhosSTOP (Roche; 04906837001) and 10 mM PR619) and rotated for 20 minutes at 4 C. After centrifugation at 17,000 g for 10 min, the supernatant was precleared using Protein G Sepharose beads for 30 min at 4 C. After that, the precleared supernatant was incubated with FLAG beads (Sigma-Aldrich; A2220) overnight. .. Beads were washed 4 times using IP buffer and eluted with 23 SDS-Loading buffer for 10 minutes at 95 C. For complex II IP, MEFs and L929 cells were stimulated as indicated in the figure legends and cells were lysed in 1% Triton X-100 IP lysis buffer (30 mM Tris-HCl [pH 7.4], 120mM NaCl, 2 mMEDTA, 2mMKCl, 1%Triton X-100, complete protease-inhibitor cocktail) on ice for 20min and centrifuged at 17,000 g for 10 min. Supernatant was precleared using Protein G Sepharose beads for 30 min at 4 C. 25 mL protein G beads were blocked for 1 h with lysis buffer containing 1%BSA and boundwith 1.5 mg FADD antibody (Santa Cruz Biotechnology; sc-6036) for 2 h and incubated with lysates for 4 h at 4 C or lysates were incubated with 3 mg FADD antibody (Merck Millipore; 05-486) for 6-8 h and then incubated with 25 mL protein G beads blocked for 1 h with lysis buffer containing 1%BSA overnight. ..

Protease Inhibitor:

Article Title: The Autophagy-Initiating Kinase ULK1 Controls RIPK1-Mediated Cell Death.
Article Snippet: Cells were lysed in 0.5%Nonidet P-40/IGEPAL CA-630 IP lysis buffer (20 mM Tris-HCl, [pH 7.4], 150 mM NaCl, 0.5% Nonidet P-40/IGEPAL CA-630, 10% glycerol, 1 3 complete protease inhibitor Cocktail (Sigma-Aldrich; P2714), 1 3 PhosSTOP (Roche; 04906837001) and 10 mM PR619) and rotated for 20 minutes at 4 C. After centrifugation at 17,000 g for 10 min, the supernatant was precleared using Protein G Sepharose beads for 30 min at 4 C. After that, the precleared supernatant was incubated with FLAG beads (Sigma-Aldrich; A2220) overnight. .. Beads were washed 4 times using IP buffer and eluted with 23 SDS-Loading buffer for 10 minutes at 95 C. For complex II IP, MEFs and L929 cells were stimulated as indicated in the figure legends and cells were lysed in 1% Triton X-100 IP lysis buffer (30 mM Tris-HCl [pH 7.4], 120mM NaCl, 2 mMEDTA, 2mMKCl, 1%Triton X-100, complete protease-inhibitor cocktail) on ice for 20min and centrifuged at 17,000 g for 10 min. Supernatant was precleared using Protein G Sepharose beads for 30 min at 4 C. 25 mL protein G beads were blocked for 1 h with lysis buffer containing 1%BSA and boundwith 1.5 mg FADD antibody (Santa Cruz Biotechnology; sc-6036) for 2 h and incubated with lysates for 4 h at 4 C or lysates were incubated with 3 mg FADD antibody (Merck Millipore; 05-486) for 6-8 h and then incubated with 25 mL protein G beads blocked for 1 h with lysis buffer containing 1%BSA overnight. ..



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NLK deficiency disrupts PANoptosome assembly and augments RIPK1/3‐ dependent necrosome formation in vivo. (A, B) Representative immunoblots of <t>FADD‐</t> 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 <t>for</t> <t>CD68</t> (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.
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Expression <t>of</t> <t>FLIP</t> and expression and phosphorylation of PKR and <t>FADD</t> 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
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Expression <t>of</t> <t>FLIP</t> and expression and phosphorylation of PKR and <t>FADD</t> 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
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Expression <t>of</t> <t>FLIP</t> and expression and phosphorylation of PKR and <t>FADD</t> 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
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Image Search Results


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.

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

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.

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

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

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