Total cell lysates were prepared in NP-40 lysis buffer as described (48 ). Soluble proteins were subjected to immunoprecipitation with anti-MUC1-C (49 (link)). Precipitates and cell lysates were analyzed by immunoblotting with anti-MUC1-C (49 (link)), anti-β-actin (Sigma), anti-TAK1 (Cell Signaling Technology), anti-phospho-IKKβ, anti-IKKβ, anti-phospho-NF-κB p65 (Cell Signaling Technology), anti-NF-κB p65 (Santa Cruz Biotechnology), anti-TRAF6, anti-BCL-XL and anti-AXIN2 (Cell Signaling Technology). Immune complexes were detected using horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence (GE Healthcare).
Anti tak1
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-TAK1 is a primary antibody that specifically recognizes the TAK1 protein. TAK1 is a serine/threonine protein kinase involved in various cellular signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Anti ikkβ, by Cell Signaling Technology (5 mentions)
Anti β actin, by Merck Group (4 mentions)
Anti nf κb p65, by Santa Cruz Biotechnology (3 mentions)
Anti p tak1, by Cell Signaling Technology (3 mentions)
Anti jnk, by Cell Signaling Technology (3 mentions)
Anti pho tak1, by Cell Signaling Technology (3 mentions)
Anti gapdh, by Cell Signaling Technology (3 mentions)
Anti p65, by Cell Signaling Technology (3 mentions)
Anti p38, by Cell Signaling Technology (3 mentions)
Anti iκbα, by Cell Signaling Technology (3 mentions)
Enhanced chemiluminescence, by GE Healthcare (2 mentions)
Anti axin2, by Cell Signaling Technology (2 mentions)
Anti phospho ikkβ, by Cell Signaling Technology (2 mentions)
Horseradish peroxidase conjugated secondary antibodies and, by GE Healthcare (2 mentions)
Anti phospho nf κb p65, by Cell Signaling Technology (2 mentions)
Anti p jnk, by Cell Signaling Technology (2 mentions)
Anti myc, by Cell Signaling Technology (2 mentions)
Anti pho ikkβ, by Cell Signaling Technology (2 mentions)
Anti ikkα β, by Cell Signaling Technology (2 mentions)
Anti caspase 3, by Cell Signaling Technology (2 mentions)
21 protocols using anti tak1
1
MUC1-C Immunoprecipitation and Immunoblotting
2
Cell Isolation and Characterization Protocol
3
Antibody-based Western Blotting and Immunoprecipitation
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Antibodies used for Western blotting included anti-Myc, anti-Flag, anti-HA, anti-pJNK, anti-JNK, anti-pIKK, anti-TAK1, anti-pTAK1, cleaved caspase 3, and anti-β-tubulin from Cell Signaling. Anti-Fli1 and anti-iNOS were purchased from Santacruz Biotechnology, and anti-TRB3 antibodies were a gift from Dr Marc Montminy, Salk Institute. For immunofluorescence, mouse anti-BAX clone 6A7 (BD Bioscience), sheep anti-insulin (Binding Site), and In Situ Cell Death Detection Kit (Fluorescein) from Roche were purchased from commercial sources. Agarose-conjugated antibodies used for immunoprecipitations included anti-HA agarose (Thermo Fisher Scientific) and anti-FlagM2 agarose (Sigma-Aldrich). Streptavidin-Agarose was purchased from Pierce. Immunofluorescence and Western blotting employed fluorescent or HRP-conjugated secondary antibodies (Jackson ImmunoResearch), the latter detected using Supersignal West-Pico Plus chemiluminescence reagent (Thermo Fisher). Other reagents include IL-1β (Peprotech) and ultrapure TLR4-specific LPS (InvivoGen). Flag-TRB3 adenovirus was generated as described 3). Smartpool TARGETplus siRNA was used to knockdown Fli1, and nontargeting siRNA was used as control (Dharmacon-Horizon Discovery Sciences). TPCA-1 (Bio-Techne-Tocris) was used to inhibit the NFκB pathway, and MLK3 was inhibited using CEP11004 (gift from Cephalon Inc).
4
Investigating p62 Signaling Pathways
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Cells were transfected with the appropriate vectors, as indicated in each figure. Western blotting and IP assays were performed as described previously (25 (link)26 (link)27 (link)28 (link)). HEK293T cells were transfected with a mock vector as a control vector and appropriate vectors. At 38 h post-transfection, the transfected cells were extracted and the cell lysates were subjected to immunoprecipitation with anti-Flag or anti-Myc antibodies followed by immunoblotting (IB) using anti-Flag, anti-Myc, anti-HA, anti-p62, anti-TRAF6, or anti-ECSIT antibodies. WT p62+/+ MEF and p62−/− MEF cells were stimulated without or with LPS for different times, lysed in lysis buffer, and the lysates were examined by Western blotting with anti-p62, anti-IKKαβ, anti-pho-IKKβ, anti-pho-TAK1, anti-TAK1, and anti-GAPDH antibodies (Cell Signaling Technology).
5
Western Blot Analysis of Protein Expression
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Total proteins in cells were collected using cell lysis buffer (Beyotime Biotechnology). Bradford Protein Assay Kit (Beyotime Biotechnology) was used for measuring proteins concentration. Western blotting was conducted using a similar method described in the earlier literature. Anti-proliferating cell nuclear antigen (PCNA) antibody (#13110), anti-p21 antibody (#2947), anti-SOX2 antibody (#3579), anti-N-cadherin (N-cad) antibody (#13116), anti-P65 antibody (#8242), anti-P65S536 antibody (#3033), anti-TAK1 (#5206), anti-phospho-IKKβ (ser180, #2697), anti-IKKβ (#8943) and anti-GAPDH antibody (#5174) were supplied by Cell Signaling Technology (MA, USA). Anti-TRAF6 antibody (ab33915), anti-vascular cell adhesion molecule-1 (VCAM1) antibody (ab134047), anti-CD44 antibody (ab189524) and anti-matrix metalloproteinase 14 (MMP14) antibody (ab51074) were supplied by Abcam Biotechnology (MA, USA). Signals of proteins were visualized through enhanced chemiluminescence technique. The relative protein expressions were calculated using Image J software.
6
Investigating GPR120 Agonists and Antagonists
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GPR120 agonists GW9508 and docosahexaenoic acid (DHA) were obtained from Sigma-Aldrich (St. Louis, MO, USA); GPR120 antagonist AH7614 was from Tocris Bioscience (Ellisville, MO, USA). Anti-GPR120 antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA); anti-Bax, anti-iNOS, anti-MCP-1, anti-NF-κB antibodies were from Abcam (Cambridge, UK). Anti-caspase 3, anit-IL-1β, anti-phospho-NF-κB, anti-PAPR, anti-phospho-γ-H2AX, anti-TAK1 (transforming growth factor-β-activated kinase 1), anti-phospho-TAK1, and anti-TNF-α antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA) and anti-β-actin were from EMD Millipore (Danvers, MA, USA). Anti-NLRP3 and anti-IL-6 were form Protein Technologies (Tucson, AZ, USA) and Biorbyt Ltd. (Taipei, Taiwan), respectively.
7
Quantitative Western Blot Analysis
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Proteins extracted from cells or tissues were quantified using Bradford Assay (Bio-Rad). Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto PVDF membranes (Bio-Rad). After blocking, the membranes were probed with primary antibodies and then incubated with specific horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology). Immunodetection was performed using enhanced chemiluminescence consistent with the manufacturer’s protocol (Thermo Fisher Scientific). Primary antibodies including anti-TAK1, anti-p-TAK1 (T187), anti-JNK, anti-p-JNK, anti-p38, anti-p-p38, anti-Smad3, anti-p-Smad3, anti-p65 and anti-β-actin were purchased from Cell Signaling Technology. Anti-p-TAK1 antibody (T184) was purchased from Thermo Fisher Scientific. Anti-Col I, Anti-Col III and anti-SIRT1 were purchased from Abcam. Anti-p20 antibody was purchased from BioVision (Milpitas, CA, USA). The band intensities were quantified and normalized to the corresponding controls. β-Actin was used as a loading control for internal correction.
8
Antibody Validation for Signaling Pathway
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Specific anti-HA, anti-Flag anti-Myc, anti-TRAF6, anti-pho-TAK1, anti-TAK1, anti-pho-IKKβ, anti-IKKαβ, and anti-GAPDH antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). The anti-p62 and anti-ECSIT antibodies were purchased from Abcam (Cambridge, MA, USA). Mouse TrueBlot ULTRA: anti-Mouse Ig HRP was purchased from Rockland Immunochemicals, Inc. (Limerick, PA, USA).
9
Signaling Pathway Characterization Protocol
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Anti–TAK‐1 (catalog no. D94D7), anti–MKK‐4 (catalog no. 9152S), anti–phospho–MKK‐4T261 (catalog no. 9151S), anti‐IκB (catalog no. 4814S), anti–phospho‐IκB, Ser32/36 (catalog no. 9246), anti–phospho–p38‐T180/Y182 (catalog no. 9211S), anti–phospho–TAK‐1‐T184/187 (catalog no. 45085), anti–phospho–IKKα/β‐Ser176/180 (catalog no. 16A6), and anti–phospho–activating transcription factor 2 (ATF‐2)–Thr71 (catalog no. 9221) were from Cell Signaling Technology. Anti–phospho–JNK‐pTpY183/185 (catalog no. 44682G) was obtained from Invitrogen. Anti–T‐ERK (catalog no. sc‐94), anti–NF‐κB–p65 (catalog no. Sc‐372), Anti–TAK‐1 (catalog no. sc‐7162), and anti–phospho–TAK‐1‐S192 (catalog no. sc‐130219) were from Santa Cruz Biotechnology. Anti‐ubiquitin–Lys‐63–specific antibody (catalog no. 05‐1308) was from Millipore. Myelin basic protein (MBP; catalog no. M1891) and anti–phospho‐ERK (catalog no. M9692) were from Sigma. A ubiquitin chain restriction enzyme analysis (UbiCREST) kit (catalog no. K‐400) was obtained from R&D Systems, and γ32P‐ATP was obtained from PerkinElmer. TAK‐1 inhibitor 5z‐7‐oxozeanol (catalog no. 3604) was from Tocris.
10
Western Blot Analysis of NF-κB Signaling
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Lysates were subject to gel electrophoresis using 5–12% Bis-Tris gels (Invitrogen), transferred to optitran nitrocellulaose membranes (Sigma) and blocked in TBS-0.1% tween with 5% non-fat dried milk. Primary antibodies were probed overnight in TBS-0.1% Tween containing 5% BSA with secondary antibodies probed in TBS-0.1% Tween containing 5% non-fat dried milk for 1 h. Immunoblots were developed using ECL prime (GE Life Sciences) on CL-XPosure film (Sigma) using an EcomaxProtec developer (Photon Imaging Systems). See supplementary materials and methods for extended protocol including cell lysis. Antibodies list: anti-P-p65 ser536 (#3031), anti-P-IKKα/β (#2697), anti-IKKβ (#8943), anti-P-TAK1 (#4508) and anti-TAK1 (#5206) were purchased from Cell Signalling Technology. Anti-p65 (Santa Cruz Biotechnology: sc-372), Anti-P-p65 ser529 (eBioscience: 14-9864-82), anti-P-p65 ser276 (Millipore: AB3375), anti-GAPDH (Sigma: G9295) and anti-β-Actin (Sigma: A1978). Goat anti-rabbit (P0448) and goat anti-mouse (P0447) secondary antibodies were purchased from Dako.
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