PE was purchased from Tokyo Chemical Industry (P0398). The autophagy inhibitor CQ was obtained from Sigma‐Aldrich, St Louis, MO, USA (C6628) and was applied to cardiomyocytes at a concentration of 10 μM 16 . Protein G‐Agarose was purchased from Roche (Roche Diagnostics, Mannheim, Germany). For the Western blotting detection of specific proteins, the following primary antibodies obtained from Cell Signaling Technology (Danvers, MA, USA) were used: anti‐Atg7 (#2631), anti‐AMPK (Thr172) (#2531), anti‐AMPK (#2532), antiphosphorylated mTOR (Ser2448) (#5536), antiphosphorylated p70S6K (Thr389) (#9234), anti‐p70S6K (#2708), antiphosphorylated Akt (Ser473) (#4058), anti‐Akt (#9272) and anti‐GAPDH (#2118) antibodies. Anti‐Sestrin 1 antibody was obtained from Abcam, Cambridge, UK (ab134091). Anti‐LC3B antibody was obtained from Novus Biologicals, Littleton, CO, USA (NB100‐2220). Anti‐p62 antibody was purchased from Sigma‐Aldrich (P0068). Antivinculin antibody was purchased from Sigma‐Aldrich (V9264). Anti‐cathepsin D antibody was obtained from Santa Cruz Biotechnology, Dallas, TX, USA (sc‐6486).
Anti phosphorylated mtorser2448
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-phosphorylated (p) mTORser2448 is a laboratory reagent that detects the phosphorylation of the mammalian target of rapamycin (mTOR) protein at serine 2448. The phosphorylation of mTOR at this site is a key indicator of mTOR activation, which is involved in cellular processes such as cell growth, proliferation, and metabolism.
Automatically generated - may contain errors
Lab products found in correlation
Anti mtor, by Cell Signaling Technology (4 mentions)
Antiphosphorylated akt ser473, by Cell Signaling Technology (2 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti p70s6k, by Cell Signaling Technology (2 mentions)
Anti vinculin antibody, by Merck Group (2 mentions)
Anti ampk, by Cell Signaling Technology (2 mentions)
Anti atg7, by Cell Signaling Technology (1 mentions)
Anti ampk thr172, by Cell Signaling Technology (1 mentions)
Antiphosphorylated p70s6k thr389, by Cell Signaling Technology (1 mentions)
Anti lc3b antibody, by Novus Biologicals (1 mentions)
Anti p62 antibody, by Merck Group (1 mentions)
Anti cathepsin d antibody, by Santa Cruz Biotechnology (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Protein g agarose, by Roche (1 mentions)
Anti p62, by Cell Signaling Technology (1 mentions)
Anti phosphorylated p38, by Cell Signaling Technology (1 mentions)
Anti phosphorylated jnk, by Cell Signaling Technology (1 mentions)
Anti phosphorylated erk1 2, by Cell Signaling Technology (1 mentions)
Anti lc3, by Cell Signaling Technology (1 mentions)
Anti nlrp3, by Cell Signaling Technology (1 mentions)
5 protocols using anti phosphorylated mtorser2448
1
Autophagy Modulation in Cardiomyocytes
2
NLRP3 Inflammasome Regulation by Guttiferone K
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RIPA lysis buffer, BCA Protein Assay Kit, and Protein A/G agarose/sepharose beads were obtained from the Beyotime Institute of Biotechnology (Shanghai, China). The following antibodies were used: anti-NLRP3, anti-IL-1β, anti-ASC, anti-LC3, anti-p62, anti-Akt, anti-phosphorylated Akt (Ser473), anti-mTOR, anti-phosphorylated mTOR (Ser2448), anti-phosphorylated p38, anti-phosphorylated JNK, and anti-phosphorylated ERK1/2 were purchased from Cell Signaling Technology, Inc. (CST, Danvers, MA, USA); anti-IL-1β was purchased from R&D Systems (Minnesota, USA); BECN1 siRNA, Transfection Reagents, rabbit anti-caspase-1, goat anti-rabbit, donkey anti-goat, and goat anti-mouse LC3 were purchased from Santa Cruz Biotechnology, Inc.; anti-β-actin monoclonal antibody was from ProteinTech Group (Chicago, IL); goat anti-NLRP3 was purchased from Abcam (Cambridge, UK). Guttiferone K (GK, purity > 98%, C38H50O6, MW: 602.80) was isolated from Garcinia yunnanensis as previously described [29 (link)]; Dulbecco's Modified Eagle's Medium (DMEM) was obtained from HyClone Laboratories, Inc. (Logan, UT, USA). Middlebrook 7H9 and 7H10 media were obtained from Difco (Detroit, MI, USA), and oleic acid-albumin-dextrose-catalase (OADC) supplements were from BD Biosciences (BD, Sparks, MD, USA).
3
Comprehensive Immunoblot Analysis of mTOR Pathway
4
Western Blot Analysis of Cellular Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were lysed with RIPA buffer as described previously (27 (link)), and proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The membranes were blocked with bovine serum albumin and incubated with the following primary antibodies at 4°C overnight: anti-MAP3K7 (4505, Cell Signaling Technology, Danvers, MA), anti-phosphorylated (p)-mTOR (Ser2448) (5536, Cell Signaling Technology), anti-mTOR (2983, Cell Signaling Technology), anti-p-AMPK (5536, Cell Signaling Technology), anti-AMPK (2532, Cell Signaling Technology), anti-p-Ser792-raptor (2083, Cell Signaling Technology), anti-raptor (2280, Cell Signaling Technology), and anti-ACTB (A5441, Sigma-Aldrich, St. Louis, MO). The proteins were probed with IRDye-800 or−680 secondary antibodies (LI-COR, Lincoln, NE) at room temperature for 1–2 h and scanned to analyze protein expression with an Odyssey® Imaging System (LI-COR).
5
Western Blot Analysis of mTOR Pathway
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted from kidney tissues using RIPA lysis buffer as previously described (23 (link)). The supernatant was collected, and the concentration was determined using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). Then, protein samples (40-60 µg) were separated using 8-10% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were then blocked in PBS containing 3% BSA and incubated overnight at 4˚C with appropriate primary antibodies, including anti-mTOR (1:1,000; Cell Signaling Technology, Inc.), anti-phosphorylated (p-)mTOR (Ser 2448, 1:1,000; Cell Signaling Technology, Inc.), anti-p70S6K (1:500; Cell Signaling Technology, Inc.), anti-p-p70S6K (Ser 371, 1:500; Cell Signaling Technology, Inc.), anti-Deptor (1:800; Cell Signaling Technology, Inc.) and anti-α-SMA (1:1,000; Abcam). Near-infrared fluorescence-conjugated secondary antibodies (1:10,000; LI-COR) were used to detect the proteins at room temperature and were developed colorimetrically using the Odyssey biocolor infrared fluorescence imaging system (LI-COR). Quantification was performed by measuring the signal intensity using the ImageJ software.
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!