Cells were seeded at appropriate density 1 day before drug treatment. Cells stimulated by EGF were starved with serum-free culture medium overnight. Various inhibitors (taken from 20 mM stock in DMSO) were first prepared as 100x concentrated solutions before being added to cell culture. Cells were treated for 24 h or as indicated, lysed in NuPAGE-LDS lysis buffer (Invitrogen) and immunoblotted with primary antibodies against TF (R&D, Cat#MAB2339), P-EGFR (Epitomics, Cat#1138-1), Raptor (Proteintech, Cat#20984-1-AP), Rictor (CST, Cat#2114), P-S6 (Epitomics, Cat#2268-1), P-AKT (Abcam, Cat#Ab81283), pERK (CST, Cat#4370), and GAPDH (Bioworld, Cat#AP0063).
Raptor
Manufactured by Proteintech
Sourced in United States
The Raptor is a high-performance liquid chromatography (HPLC) system designed for efficient and reliable analysis of a wide range of biomolecules. It features a modular design, allowing for customization to suit specific research or analytical needs. The Raptor provides precise control over flow rates, gradients, and column temperature to ensure accurate and reproducible results.
Automatically generated - may contain errors
Lab products found in correlation
P akt, by Abcam (1 mentions)
P egfr, by Abcam (1 mentions)
Nupage lds lysis buffer, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Bioworld Technology (1 mentions)
β catenin 8480, by Cell Signaling Technology (1 mentions)
P ampk α 2535, by Cell Signaling Technology (1 mentions)
Cleaved caspase 7, by Cell Signaling Technology (1 mentions)
Hif 1α, by Novus Biologicals (1 mentions)
Pd l1, by Cell Signaling Technology (1 mentions)
Anti pimonidazole, by Hypoxyprobe (1 mentions)
Cyclin d3 2936, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Complete protease inhibitor cocktail, by Merck Group (1 mentions)
α tubulin, by Proteintech (1 mentions)
Itgb4, by Proteintech (1 mentions)
Lumiglo peroxidase chemiluminescent substrate kit, by LGC (1 mentions)
Mab374 antibody, by Merck Group (1 mentions)
C myc, by Santa Cruz Biotechnology (1 mentions)
Immun blot pvdf membrane, by Bio-Rad (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
4 protocols using raptor
1
Investigating EGF-Mediated Signaling Pathways
2
Antibodies and Their Sources
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies were obtained from venders as follows: HIF-1α (Novus biologicals. 10771 E Easter Ave, Centennial, CO, USA. NB100-479), Raptor (Proteintech. 5500 Pearl St # 400, Rosemont, IL, USA. 20984-1-AP), anti-pimonidazole (Hypoxyprobe. 121 Middlesex Turnpike, Burlington, MA, USA. 4.3.11.3 mouse MAb), septin-2 (Novus biologicals, NBP2-75660), septin-5 (Proteintech, 11631-1-AP), septin-11 (Proteintech, 14672-1-AP). Other septin antibodies were purchased from Sigma Aldrich (septin-6, HPA005665; septin-7, HPA029524; septin-9, HPA042564; septin-10, HPA056304). All other antibodies were purchased from Cell Signaling Technology, (3 Trask Lane, Danvers, MA, USA): AMPKα (5831), p-AMPKα (2535), p-Raptor (2083), ACC (3676), p-ACC (11818), α-tubulin (2144), acetyl-α-tubulin (5335), β-catenin (8480), HER2 (4290), p-HER2 (2247), Erk1/2 (9102), p-Erk1/2 (4370), cleaved PARP (5625), cleaved caspase 7 (9491), and PD-L1 (13684).
3
Western Blot Analysis of Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The seven-day cultured SCs were washed with cold phosphate-buffered saline (PBS) and lysed in RIPA buffer (20 to 188, EMD Millipore, USA) containing cOmplete protease inhibitor cocktail (EMD Millipore). Cell lysates were separated by SDS-PAGE followed by western blotting with the indicated antibodies (ATF2: 14834 to 1-AP, Raptor: 20984 to 1-AP, IKBKG: 18474 to 1-AP; α-Tubulin: 11224 to 1-AP; Proteintech, USA) (Cyclin D3:2936; Cell Signaling Technology, USA). Western blot analyses were performed as described previously.29 (link)
4
Western Blotting for Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein extracts were prepared according to previously published protocol (27 (link)). Briefly 5 μL of protein lysates were loaded onto SDS-polyacrylamide gels and transferred to Immun-Blot PVDF membranes (Bio-Rad Laboratories). After blocking in 5% milk, the membranes were incubated in primary antibodies against the following: p63 (4A4, 1:20,000), ΔNp63 (E6Q3O; Cell Signaling Technology, 1:5000), ITGB1 (Proteintech, 1:10,000), ITGB4 (Proteintech, 1:10,000), cMYC (Santa Cruz Biotechnology, 1:5000), AKT1 (Proteintech, 1:10,000), mTOR (Proteintech, 1:10,000), Raptor (Proteintech, 1:10,000), S6 (Cell Signaling Technology, 1:5000), and p-S6 (Cell Signaling Technology, 1:5000). The MAB374 antibody (EMD Millipore) was used to detect GAPDH as a loading control at 1:20,000 dilution. HRP-conjugated secondary antibodies corresponding to the primary antibody host were incubated with each blot. Unbound antibodies were washed off in 0.05% Tween-20 in Tris-buffered saline. The LumiGLO peroxidase chemiluminescent substrate kit (SeraCare) was used to detect antibody-labeled proteins, and membranes were imaged using the Bio-Rad ChemiDoc imaging system.
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!