Western blotting experiment was described previously (Zhao et al., 2019 (link)), and the detection of RIPK3 or MLKL oligomers was performed according to the method described by Wang et al. (2019) (link). The following antibodies were used for the experiments: anti-FADD (1:1000, 610399) and anti-RIPK1 (1:3000, 610458) (BD Transduction Laboratories, San Jose, CA); anti-phospho-RIPK3 (1:1000, ab195117), anti-MLKL (1:1000, ab172868), anti-phospho-MLKL (1:1000, ab196436) (Abcam, Cambridge, MA, United States); anti-RIPK1 (1:3000, 3493), anti-RIPK3 (1:3000, 15828), anti-Myc tag (1:2000, 2276), anti-HA tag (1:2000, 3724), anti-Flag tag (1:2000, 14793), anti-TRAF2 (1:1000, 4724), anti-phospho-RIPK1 (1:1500, 31122) and anti-phospho-RIPK3 (1:1000, 57220) [Cell Signaling Technology (CST), Beverly, MA]; anti-TRADD (1:1000, ABP52634) and anti-GAPDH (1:1500, ABP52783) (Abbkine, Redlands, CA, United States); anti-β-actin (1:3000, A5441) (Sigma-Aldrich). All western blot assays were performed three times, and the representative results are shown.
Anti ripk1
Manufactured by BD
Sourced in United States
Anti-RIPK1 is a laboratory tool used for the detection and quantification of RIPK1 (Receptor-Interacting Serine/Threonine-Protein Kinase 1) in biological samples. It functions as a research reagent to assist in the study of RIPK1-related cellular processes and signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Anti ripk3, by ProSci (7 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (4 mentions)
Anti phospho mlkl, by Abcam (3 mentions)
Anti β actin, by Merck Group (3 mentions)
Anti mlkl, by Merck Group (3 mentions)
Anti cd9, by System Biosciences (3 mentions)
Anti cd63, by System Biosciences (3 mentions)
Anti cd81, by System Biosciences (3 mentions)
Ab196436, by Abcam (2 mentions)
Anti ripk3, by Cell Signaling Technology (2 mentions)
Anti phospho ripk1, by Cell Signaling Technology (2 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti mtor, by Cell Signaling Technology (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti ripk3, by Abcam (2 mentions)
Ciap1 2, by R&D Systems (2 mentions)
Anti cleaved parp, by Cell Signaling Technology (2 mentions)
Anti fadd, by Abcam (2 mentions)
Anti p mlkl, by Abcam (2 mentions)
Nec 1, by Merck Group (2 mentions)
26 protocols using anti ripk1
1
Detecting RIPK3 and MLKL Oligomers
2
Inhibition of Akt and Necroptosis Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following materials were purchased from commercial companies: TNFα (PeroTech; Rocky Hill, NJ, USA); pan-caspase inhibitor z-VAD-fmk (Abcam, Cambridge, MA, USA). InSolution Akt Inhibitor viii isozyme-selective, Akti-1/2 and InSolution rapamycin were obtained from Calbiochem (San Diego, CA, USA). MitoSox Red was obtained from Invitrogen (Carlsbad, CA, USA). Hoechst 33258, butylated hydroxyanisole (BHA) and rotenone were obtained from Sigma (St. Louis, MO, USA). Nec-1 (5-(7-chloro-1H-indol-3-ylmethyl)-3-methylimidazolidine-2,4-dione), the inactive analog of necrostatin-1 analog (Nec-1i; 5-(7-chloro-1H-indol-3-ylmethyl)imidazolidine-2,4-dione),3 (link), 12 (link), 13 (link) was a kind gift from Dr. Greg Cuny. LOX-1 was obtained from Chembridge (compound 5680672; San Diego, CA, USA), and Bai was from Cayman Chemicals (Ann Arbor, MI, USA). Antibodies were obtained from commercial sources: anti-pAkt-473, anti-p–Akt-308, anti-p-GSK-3β, anti-p-mTOR, anti-p-S6, anti-mTOR, anti-Akt1, anti-Akt2, anti-Akt3, and anti-Akt from Cell Signaling (Danvers, MA, USA); anti-RIPK1 from BD Transduction Laboratories (Lexington, KY, USA); anti-RIPK3 from ProSci Incorporated (San Diego, CA, USA); anti-HMGB1 and β-actin were obtained from Abcam.
3
Immunoprecipitation of RIPK1 from Cell Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Briefly, cells were collected with a cell scraper and centrifuged at 5000 × g for 5 min. The pellet was re-suspended in RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 μg/ml PMSF) with phosphatase and protease inhibitor cocktail and stored at − 80 °C. Protein levels were quantified with Pierce BCA Protein Assay Kit (Thermo Scientific). Immunoprecipitation experiments were performed by incubating 100 μg of protein with 2.5 μg of anti-RIPK1 (BD Transduction Laboratories) with rotation at 4 °C for 48 h. Then, 50 μl of protein G magnetic beads (Biorad) were added to each sample and incubated with rotation at 4 °C for 3 h. Following magnetic separation, beads were mixed with loading buffer and boiled at 90 °C for 5 min. After the elution step, samples were analyzed by western blotting as described.
4
ChIP-qPCR Analysis of Protein Binding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ChIP analyses were performed as previously described [30 (link)]. In brief, quantitative PCR was performed using a THUNDERBIRD® qPCR Mix (TOYOBO) and the StepOne real-time PCR system (Applied Biosystems) with the primer listed in Supplementary Table S3 . The antibodies for ChIP analysis used in this study were anti-Flag M2 (F3165, Sigma), anti-RAR (M-454, Santa Cruz), and anti-RIPK1 (610458, BD Transduction Laboratories). At least three biological experiments were carried out and data are presented as means ± SD.
5
Western Blot Analysis of Necroptosis Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RIPA Lysis Buffer (Beyotime, China) containing PMSF (Beyotime, China) and PhosSTOP (Sigma-Aldrich, USA) was used to lyse the cells with indicated treatment. After quantification, the protein samples were separated by SDA-PAGE gel (Beyotime, China). Primary antibodies (Anti-RIPK1, BD Biosciences, USA; Anti-RIPK3, Abcam, USA; p-RIPK3, Abcam, USA; Anti-MLKL, Sigma-Aldrich, USA; Anti-p-MLKL, Sigma-Aldrich, USA; Anti-GAPDH, Beyotime, China) were used at 4 °C overnight. The protein bands were detected by ECL Kit (BOSTER, China).
6
Western Blot Analysis of Cell Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole-cell extracts were prepared from cells using RIPA lysis buffer. Western blotting analysis was performed using anti-RIPK1 (BD Biosciences, San Jose, CA, USA), RIPK3, p21 (Santa Cruz), cIAP1/2 (R&D Systems, Minneapolis, MN, USA), PARP-1 (BD Biosciences) and cleaved PARP-1 (Cell Signaling Technology, Danvers, MA, USA) antibodies. Anti-HSP90 (BD Biosciences) and β-actin (Prosci, Poway, CA, USA) antibodies were used as a loading control.
7
RIPK3 and Caspase-8 Immunoprecipitation
8
Quantification of Cell Death Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To analyze cell death pathways induced in ESCRT-III-silenced cells, total (live and dead) supernatant and adherent cells were collected and counted. 1 × 106 cells were pelleted and lysed in 100 μL 2XSB buffer without DTT (4% SDS (#EU0660, Euromedex), 0.12 M Tris pH 6,8 (#10708976001, Sigma-Aldrich), glycerol 10% (#24388.295, VWR), dash of bromophenol blue (#B-5525, Sigma-Aldrich)) (1 million cells lysed per 100 μL buffer). When needed, DTT (#P2325, ThermoFisher) was added extemporaneously at 100 mM final concentration. Samples were immediately boiled for 5 min at 95°C, quickly spun and frozen at −20°C until use. The following antibodies were used for immunoblotting detection: anti-caspase-3 (#9662, Cell Signaling Technologies), anti-caspase-8 (#9429, Cell Signaling Technologies), anti-caspase-1 (produced in house – Peter Vandenabeele’s lab and Schotte et al. (2004) (link), anti-caspase-11 (#120–10454, Novus Biologicals), anti-cleaved Parp (#9544, Cell Signaling Technologies), anti-Gasdermin-D (Genentech), anti-IL-1β (#GTX74034, Genetex), anti MLKL (#MABC604, Millipore (reducing conditions) or #SAB1302339, Sigma-Aldrich (non-reducing conditions)), anti-phosphoMLKL (#ab196436, Abcam), anti-RIPK3 (#R4277, Sigma-Aldrich), anti-RIPK1 (#610459, BD Biosciences).
9
Western Blot Analysis of Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following primary antibodies were used for western blots of both mouse and human cell lines: anti-MLKL (produced in-house;25 (link) available as MABC604, EMD Millipore, Billerica, MA, USA), anti-GAPDH (2118, Cell Signalling Technology, Danvers, MA, USA), anti-Actin (A-1987, Sigma-Aldrich, St Louis, MO, USA), anti-RIPK1 (610458, BD Biosciences, Franklin, NJ, USA), anti-Hsp90 (ADI-SPA-835, Enzo, Life Sciences, Farmingdale, NY, USA), anti-Cdc37 (D11A3, Cell Signalling Technology), and anti-VDAC (Sigma-Aldrich). Anti-mouse RIPK3 (PSC-2283-c100, Axxora, San Diego, CA, USA) and anti-human RIPK3 (ab56164, Abcam, Cambridge, UK) were used for their respective cell lines.
Recombinant hTNF-Fc, produced in-house, and the Smac mimetic, Compound A, were described previously.71 (link), 72 (link) QVD-OPh was obtained from R&D Systems (Minneapolis, MN, USA). Both NVP-BEP800 and 17-AAG were obtained from Selleckchem (Sydney, NSW, Australia), and AT13387 was produced by Active Biochem (Wanchai, Hong Kong).
Recombinant hTNF-Fc, produced in-house, and the Smac mimetic, Compound A, were described previously.71 (link), 72 (link) QVD-OPh was obtained from R&D Systems (Minneapolis, MN, USA). Both NVP-BEP800 and 17-AAG were obtained from Selleckchem (Sydney, NSW, Australia), and AT13387 was produced by Active Biochem (Wanchai, Hong Kong).
10
RIPK1 and RIPK3 Protein Complexes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Brain homogenates from a block of tissue containing the entire cerebral contusion were lysed in RIPA buffer containing protease and phosphatase inhibitors (Sigma-Aldrich), followed by sonication and centrifugation at 15,000 × g for 30 min. Supernatants containing protein complexes were incubated with anti-RIPK3 (Prosci, #2283) or anti-RIPK1 (BD bioscience, #610458) antibodies conjugated to Magnetic Protein G Dynabeads (Thermo Fisher Scientific) at 4 °C overnight. Beads were washed three times with washing buffer and proteins were eluted in 1X loading buffer and processed/ for western blot.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!