Western blotting analysis was performed as previously described [16 (link)]. Antibodies against the following proteins were used: Cleaved-caspase-3 (1:1000, #9661), PARP (1:1000, #9542), Bcl-2 (1:1000, #15071), survivin (1:1000, #2808), p-MLKL (1:1000, #91689), MLKL (1:1000, #14993), p-RIP3 (1:1000, #93654,) RIP3 (1:1000, #13526), p-RIP1 (1:1000, #65746), RIP1 (1:1000, #3493), LC3A/B (1:1000, #12741), beclin1 (1:1000, #3495), p62 (1:1000, #5114), p-PI3K (1:1000, #4228), PI3K (1:1000, #4257), p-AKT (1:1000, #4060), AKT (1:1000, #4691), p-mTOR (1:1000, #2974), mTOR (1:1000, #2983), p-p70S6K (1:1000, #9204), and p70S6K (1:1000, #2708), all from Cell Signaling Technology (Beverly, MA, USA); Cyclin D1 (1:1000, #sc-20044), β-actin (C4, 1:1000, #sc-47778), Cdk4 (1:1000, #sc-23896), and Cdk6 (1:1000, #sc-7961), all from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and MMP13 (1:1000, NBP1-45723) from Novus Biologicals (Centennial, CO, USA). Protein bands were detected using the ProteinSimple detection system (ProteinSimple Inc., Santa Clara, CA, USA).
P rip3
Manufactured by Cell Signaling Technology
Sourced in United States
P-RIP3 is a recombinant rabbit monoclonal antibody that recognizes phosphorylated RIP3 (receptor-interacting protein kinase 3). RIP3 is a key mediator of necroptosis, a form of programmed cell death.
Automatically generated - may contain errors
Lab products found in correlation
P mlkl, by Cell Signaling Technology (3 mentions)
Beclin 1, by Cell Signaling Technology (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
Survivin, by Cell Signaling Technology (2 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
β actin c4, by Santa Cruz Biotechnology (2 mentions)
P rip1, by Cell Signaling Technology (2 mentions)
Mmp 13, by Novus Biologicals (2 mentions)
Cyclin d1, by Santa Cruz Biotechnology (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
P p70s6k, by Cell Signaling Technology (1 mentions)
P70s6k, by Cell Signaling Technology (1 mentions)
Lc3a b, by Cell Signaling Technology (1 mentions)
P mtor, by Cell Signaling Technology (1 mentions)
P pi3k, by Cell Signaling Technology (1 mentions)
β actin, by ABclonal (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Bradford reagent, by Bio-Rad (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
4 protocols using p rip3
1
Western Blot Analysis of Apoptosis and Signaling
2
Western Blot Analysis of Apoptosis Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Radioimmunoprecipitation assay (RIPA) lysis buffer was used for total protein extraction and a BCA assay was applied to identify protein concentrations. Samples (40 μg each) were separated using 10-15% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with specific primary antibodies after blocking. The antibodies against p53, cyclin D1, cyclin E1, p21 WAF1/CIP1 , p27 KIP1 , caspase-3, caspase-9, caspase-8, c-FLIP, poly(ADP-ribose)polymerase 1 (PARP-1), BCL2, BAX, BAK, p-receptor-interacting serine/threonine kinase (pRIP), RIP, p-RIP3, RIP3, and p-mixed lineage kinase domain-like pseudo kinase (MLKL) were obtained from Cell Signaling Technology (CST; Danvers, USA; 1:1,000 dilution), and β-actin was obtained from ABclonal Biotech (1:5,000 dilution; Wuhan, China). The protein bands were visualized using chemiluminescent detection.
3
Western Blotting Analysis of Cell Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting was performed as described previously [61 (link)]. Briefly, the total protein concentration was determined using the Bradford reagent (Bio-Rad, Hercules, CA, USA), and 20 μg of total proteins was separated using SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA, USA). The following antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA) and used: AKT (1:1000, #4691), p-AKT (1:1000, #4060), Bax (1:1000, #2772), Bcl-2 (1:1000, #15071), Beclin1 (1:1000, #3495), Caspase-3 (1:1000, #9662), MLKL (1:1000, #14993), p-MLKL (1:1000, #91689), PARP (1:1000, #9542), p-PRAS40 (1:1000, #2997), p62 (1:1000, #5114), p-RB (1:1000, #8180), RIP (1:1000, #3493), p-RIP (1:1000, #65746), RIP3 (1:1000, #13526), pRIP3 (1:1000, #93654), p-STAT3 (1:1000, #9145), and Survivin (1:1000, #2808). β-actin (C4, 1:1000, #sc-47778), Cdk4 (1:1000, #sc-23896), Cdk6 (1:1000, #sc-7961), and CyclinD1 (1:1000, #sc-20044), were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). MMP13 (1:1000, NBP1-45723) was purchased from Novus Biologicals (Centennial, CO, USA). Protein signals were detected using the ProteinSimple detection system (ProteinSimple Inc., Santa Clara, CA, USA).
4
Western Blot Analysis of Necroptosis Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Homogenized animal tissues and treated cells were lysed with RIPA lysis buffer (Roche, Basel, Switzerland) containing protease inhibitors and phosphatase inhibitors. BCA protein assay kit (Beyotime) was used to measure protein concentrations. Proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes, which were blocked for 1 h and then blotted with specific primary antibodies against actin, GAPDH, RIP1, RIP3, MLKL, phosphor (P)-RIP1, P-RIP3, and P-MLKL (1:1000 dilution; Cell Signaling Technology, MA, USA) overnight at 4℃. The membranes were washed with TBS-T several times and further incubated with the secondary antibodies (1:1000 dilution) at 37℃ for 1 h. Finally, chemiluminescence images were developed with a SuperSignal Sensitivity Substrate kit (Thermo Fisher Scientific, MA, USA) and detected using a ChemiDoc XRS + imaging system (Bio-Rad, CA, USA).
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!