Collagenase type I, DNase I, and Pronase were purchased from Sigma Aldrich. The anti-CD133 antibody was purchased from Miltenyi Biotech. The anti-αSMA, anti-IL6, anti-pNFκB (S536), anti-NFκB, and anti-TAK1 antibodies were purchased from Abcam. The anti-β2SP antibody was a gift from Dr. Lopa Mishra's laboratory. The anti-pYSTAT3, anti-STAT3, anti-TAK1, anti-pIκK α/β and anti-pTAK1 antibodies were purchased from Cell Signaling Technology. The anti-IκKβ was purchased from R&D systems.
Anti p tak1
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-p-TAK1 is a laboratory reagent that specifically recognizes and binds to the phosphorylated form of the Transforming Growth Factor-β-Activated Kinase 1 (TAK1) protein. TAK1 is a key mediator of various signaling pathways, including those involved in inflammatory responses and cell survival. The Anti-p-TAK1 product can be used to detect and study the activation state of TAK1 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti p iκbα, by Cell Signaling Technology (4 mentions)
Anti iκbα, by Santa Cruz Biotechnology (4 mentions)
Anti tak1, by Cell Signaling Technology (3 mentions)
Anti β actin, by Merck Group (2 mentions)
Anti ikkβ, by Cell Signaling Technology (2 mentions)
Anti p ikkα β, by Cell Signaling Technology (2 mentions)
Anti p jnk, by Cell Signaling Technology (2 mentions)
Tgf β elisa kit, by R&D Systems (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Anti rela, by Cell Signaling Technology (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
M mlv rt system, by Promega (2 mentions)
Anti p rela, by Cell Signaling Technology (2 mentions)
Dnase 1, by Merck Group (1 mentions)
Collagenase type 1, by Merck Group (1 mentions)
Pronase, by Merck Group (1 mentions)
Anti α sma, by Abcam (1 mentions)
Anti tak1, by Abcam (1 mentions)
Anti py stat3, by Cell Signaling Technology (1 mentions)
9 protocols using anti p tak1
1
Cell Isolation and Characterization Protocol
2
Western Blot Analysis of Stress Signaling
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Tissue samples were homogenized in standard RIPA buffer with PMSF. Protein concentrations were quantified with the Lowry assay using the DC protein assay kit (Bio-Rad, Hercules, CA, USA). Then, 30 μg of total protein was diluted to the same volume 2 × SDS-sample buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 20% glycerol, 5% 2-mercaptoethanol, and 0.025% bromophenol blue). Cultured cells were lysed in 2 × SDS-sample buffer. Protein lysates were separated by 6–10% SDS-gel electrophoresis, and transferred onto Immobilon-P membranes (Millipore, Billerica, MA, USA). Membranes were blocked in PBST buffer containing 3% skim milk for 1 h at room temperature, probed with primary antibodies and secondary HRP-conjugated antibodies (GE Healthcare, Little Chalfont, UK), and developed using ECL western blot detection reagent (GE Healthcare). Antibodies used in this study are as follows: anti-GADD34 was from Santa Cruz; anti-β-actin was from Sigma; and anti-p-eIF2α, anti-eIF2α, anti-ATF4, anti-CHOP, anti-p-NF-κB p65, anti-NF-κB p65, anti-p-IκBα, anti-IκBα, anti-p-IKKα/β, anti-IKKβ, anti-p-TAK1, anti-p-IRF3, anti-IRF3, anti-p-IKKɛ, and anti-IKKɛ were from Cell Signaling Technology (Danvers, MA, USA).
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
Hypothalamic Protein and RNA Quantification
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Hypothalami were isolated as previously described14 (link). Tissue lysis, SDS/PAGE and Western blotting were performed as previously described19 (link). Primary antibodies in Western blots included anti-IκBα (Santa Cruz, #SC847, 1:500), anti-p-TAK1 (Cell Signaling, #4531, 1:1000), anti-p-IκBα (Cell Signaling, #2859, 1:1000), anti-RelA (Cell Signaling, #3039, 1:1000), anti-p-RelA (Cell Signaling, #4764, 1:1000), and anti-β-actin (Cell Signaling, #4967S, 1:1000), and secondary antibodies were HRP-conjugated anti-rabbit or goat antibody (Pierce, 1:2000). Quantification of Western blots was processed with Image J. Total RNA was extracted from hypothalamic tissue using TRIzol® (Invitrogen), and cDNA was synthesized using M-MLV RT System (Promega). Using SYBR® Green PCR Master Mix (Applied Biosystems), expression levels of target genes were analyzed via PCR amplification and quantification. GAPDH or TBP mRNA levels were used for normalization. CSF collection and TGFβ measurement: An anesthetized mouse was placed onto the stereotactic apparatus with the head forming an angle of about 135° with the body, and then a sagittal incision in the neck skin was made inferior to the occiput, followed by penetrating a capillary tube through the dura mater into the citerna magna to draw the CSF. Serum and CSF TGF-β content were measured using TGF-β ELISA kit (R&D Systems).
5
Hypothalamic Protein and RNA Quantification
6
Anti-inflammatory Signaling Pathway Assay
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SDS (99%) was obtained from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). All cell culture reagents were from Gibco (Stockholm, Sweden). Lipopolysaccharide (LPS) and dimethyl sulfoxide (DMSO) were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). All reagents were purchased from Beyotime Institute of Biotechnology (Nantong, China). Anti-iNOS, anti-p-TAK1, anti-P-JNK, anti-P-ERK and anti-P-p38 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti COX-2, anti-CD14, anti-TLR4, anti-NF-κB P65, anti-IκBα and anti-topoisomerase I were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and anti-α-tubulin was purchased from Sigma.
7
Western Blot Analysis of p-TAK1
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Whole cell lysates were prepared in NP-40 lysis buffer (0.5% Nonidet P-40, 20 mM Tris-HCl [pH 7.5], 150 mM NaCl) in the presence of complete protease cocktail inhibitor. Lysates were centrifuged at 4°C for 10 min at 12,000 rpm in an Eppendorf microcentrifuge to remove cellular debris. Western blot analysis was performed as previously described,31 (link) using anti-p-TAK1 (Cell Signaling Technology) and anti-TAK1 (Santa Cruz). Immunoreactive proteins were detected using an enhanced chemiluminescence ECL kit (Amersham Biosciences).
8
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.
9
Whole Cell Extraction and Fractionation Protocol for HCEC
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For whole cell extraction, the cornea epithelium from residual donor tissues or HCECs treated with TGF-β1 for the indicated time points were lysed in RIPA buffer (P0013B, Beyotime, Beijing, China) with protease inhibitor cocktail (Millipore). After centrifugation (4°C, 10 min, 12,000 g), samples were prepared for Western blot analysis.
For preparation of cytoplasmic and nuclear fraction, HCECs were treated with TGF-β1 for 24h. Nuclear and cytoplasmic proteins of HCECs were extracted using the Nuclear and Cytoplasmic Protein Extraction Kit (P0028, Beyotime, Beijing, China) according to manufacturer's instructions.
Western blot were performed as described previously [52 (link)]. The membranes were probed with the following primary antibodies: anti-GAPDH (KC-5G5, Kangchen), anti-β-actin (LK9001, Sungene Biotech), anti-Histone H3 (ab1791, Abcam), anti-p16 (ab51243, Abcam), anti-p53 (ab26, Abcam), anti-p21 (ab109520, Abcam), anti-TGF-β1 (sc146, Santa Cruz), anti-IκBα (sc847, Santa Cruz), anti-p-IκBα (#2859, Cell Signaling), anti-p65 (#4764, Cell Signaling), anti-p-p65 (#3039, Cell Signaling), anti-p-TAK1 (#4531, Cell Signaling), anti-eIF2α(sc133132, Santa Cruz), anti-p-eIF2α (sc293100, Santa Cruz) and subsequently reacted with HRP-conjugated secondary antibodies, respectively (Pierce, 1:3,000). Quantification of western blots was processed with Image J.
For preparation of cytoplasmic and nuclear fraction, HCECs were treated with TGF-β1 for 24h. Nuclear and cytoplasmic proteins of HCECs were extracted using the Nuclear and Cytoplasmic Protein Extraction Kit (P0028, Beyotime, Beijing, China) according to manufacturer's instructions.
Western blot were performed as described previously [52 (link)]. The membranes were probed with the following primary antibodies: anti-GAPDH (KC-5G5, Kangchen), anti-β-actin (LK9001, Sungene Biotech), anti-Histone H3 (ab1791, Abcam), anti-p16 (ab51243, Abcam), anti-p53 (ab26, Abcam), anti-p21 (ab109520, Abcam), anti-TGF-β1 (sc146, Santa Cruz), anti-IκBα (sc847, Santa Cruz), anti-p-IκBα (#2859, Cell Signaling), anti-p65 (#4764, Cell Signaling), anti-p-p65 (#3039, Cell Signaling), anti-p-TAK1 (#4531, Cell Signaling), anti-eIF2α(sc133132, Santa Cruz), anti-p-eIF2α (sc293100, Santa Cruz) and subsequently reacted with HRP-conjugated secondary antibodies, respectively (Pierce, 1:3,000). Quantification of western blots was processed with Image J.
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