GP17 (molecular weight = 947.154; purity > 98%) was purchased from Beijing Beina Chuanglian Biotechnology Research Institute (Beijing, China). The positive control drug NBP was obtained from CSPC NBP Pharmaceutical Co., Ltd. Triphenyltetrazolium chloride (TTC) was purchased from Sigma–Aldrich (MO, United States). Primary antibodies against p62/SQSTM1, LC3-B, BNIP3, NAMPT, SIRT1, SIRT2, MnSOD, PGC-1α, FOXO3 and p-FOXO3 were obtained from Abcam (Cambridge, UK). Primary antibodies against Hif1α and Beclin1 were obtained from Proteintech (Wuhan, China). A primary antibody against SIRT3 was obtained from Cell Signaling Technology (MA, USA). The inducer RAP and the inhibitors 3-MA and 2-ME were obtained from MedChemExpress (New Jersey, USA). The inhibitor AGK-7 was obtained from Abcam (Cambridge, UK). ELISA kits for IL-6, TNF–α, MCP-1, T-AOC and 4-HNE were acquired from HaiTai TongDa Sci Tech, Ltd. (Beijing, China).
Nampt
Manufactured by Abcam
Sourced in United Kingdom
NAMPT is a protein that is involved in the regulation of cellular metabolism. It catalyzes the rate-limiting step in the biosynthesis of nicotinamide adenine dinucleotide (NAD), an essential cofactor for various enzymatic reactions in the cell.
Automatically generated - may contain errors
Lab products found in correlation
Sirt1, by Abcam (1 mentions)
Bnip3, by Abcam (1 mentions)
Sirt2, by Abcam (1 mentions)
P foxo3, by Abcam (1 mentions)
Pgc 1α, by Abcam (1 mentions)
Mnsod, by Abcam (1 mentions)
Primary antibody against sirt3, by Cell Signaling Technology (1 mentions)
Foxo3, by Abcam (1 mentions)
Ab97051, by Abcam (1 mentions)
Ab97046, by Abcam (1 mentions)
β actin, by GE Healthcare (1 mentions)
Alpha tubulin, by GE Healthcare (1 mentions)
α tubulin, by Santa Cruz Biotechnology (1 mentions)
Ecl detection system, by GE Healthcare (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Protease phosphatase inhibitor cocktail, by Roche (1 mentions)
Pcreb creb, by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Ab652, by Abcam (1 mentions)
Trans blot turbo mini pvdf transfer pack, by Bio-Rad (1 mentions)
6 protocols using nampt
1
Molecular Mechanisms of Neuroprotection
2
Protein Extraction and Cytotoxic Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were were lysed in RIPA buffer (Tris–HCl pH 8.0 25 mM, NaCl 150 mM, NP40 1%, Sodium desoxycholate 1%, SDS 1%, Na3VO4 1 mM, EDTA 0.5 M, complete protease and phosphatase inhibitor cocktail, 2 mM) and then subjected to 3 sonication cycles during 5 seconds. The amount of protein was determined by Bradford assay using BSA (bovine serum albumin) as a standard. The primary antibodies were purchased from commercial sources as follows: á-tubulin 1:10000 (SIGMA – T9026), NANOG (ab80892 Rabbit polyclonal, Abcam); NAMPT (Anti-Visfatin antibody BETHYL A300-779A),. The secondary antibodies used were: Goat pAB to Rabbit IgG (HRP) 1:5000 (ABCAM ab97051), Rabbit pAB to Mouse IgG (HRP) 1:5000 (ABCAM ab97046).
Cytotoxic Assay MTT. 5x103 SF268 cells were seeded then treated with FK866 24 hours later. After 96 hours, cell viability was measured with MTT.
Cytotoxic Assay MTT. 5x103 SF268 cells were seeded then treated with FK866 24 hours later. After 96 hours, cell viability was measured with MTT.
3
Western Blot Analysis of NRK1/2 Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein lysates were extracted from tissues in RIPA buffer (50 mmol/l Tris pH 7.4, 1% NP40, 0.25% sodium deoxycholate, 150 mmol/l NaCl, 1 mmol/l EDTA) and protease/phosphatase inhibitor cocktail (Roche, Lewes, U.K.). Total protein concentration was quantified by Bio-Rad assay. Total proteins (25 μg) were resolved on a 12% SDS-PAGE gel and transferred onto a nitrocellulose membrane. Primary antibodies specific for NRK1/2 were generated and affinity purified by BioGenes (GmbH) Berlin, Germany and used at a 1:2000 dilution. Primary antibodies including NAMPT (Abcam, USA), β-Actin (Cell Signaling, USA, #12262) and α-Tubulin (Santa Cruz, USA, SC-5286) were all commercially available and used at a 1:1000 dilution. Secondary anti-mouse and anti-rabbit antibodies conjugated with HRP (Dako, Denmark) were added at a dilution of 1/10,000. Equal loading of protein content was verified using beta-actin and alpha-tubulin and bands visualized using ECL detection system (GE Healthcare, UK).
4
Quantifying Protein Signaling in Brain Regions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The PFC and HIP regions of the brain were immediately placed on ice after dissection. After sonication, total protein was extracted and quantified with a kit. Next, 60 μg of total protein was used for electrophoretic analysis for 2 h and transferred to a PVDF membrane at 100 V for 2 h under cooling conditions. pCREB/CREB (1:5000, Cell Signaling Technology), NAMPT (1:500, Abcam), and the membranes were first treated with GAPDH (1:10,000) overnight, followed by incubation with the secondary antibody conjugated to HRP (KangChen Bio-tech, Shanghai, China). Finally, the signal intensity was evaluated with Image J.
5
Protein Expression Analysis by SDS-PAGE
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed with RIPA lysis buffer; cell lysates were then subjected to an 8–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene difluoride membrane (Bio-Rad, USA), and probed with antibodies. The antibodies used here were as follows: NFIB (Abcam, UK, 1:1000 dilution), NAMPT (Abcam, UK, 1:1000 dilution), and GAPDH (Proteinates, China).
6
Visfatin Signaling in Spheroid Proteomics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After treating spheroids with visfatin (100 ng/mL) for 48 h, spheroids were lysed in lysis buffer. Proteins were separated on 4-20% Mini-Protean TGX Precast Protein Gels (Bio-Rad) and then transferred to Trans-Blot Turbo Mini PVDF transfer packs (Bio-Rad) using the Trans-Blot Turbo transfer system (Bio-Rad). The blots were blocked for 1 h with 0.02 M Tris-buffered saline containing 5% bovine serum albumin and 0.1% Tween 20 and then incubated overnight at 4°C with antibodies specific for MMP2 (#4022; Cell Signaling Technology), GLUT1 (ab652; Abcam), GLUT4 (#2213; Cell Signaling Technology), hypoxia-inducible factor (HIF-1; #41493; GeneTex), and visfatin (NAMPT; ab233294; Abcam). The membranes were then washed three times in Tris-buffered saline and 0.1% Tween 20 and incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated anti-rabbit (#7074) or anti-mouse (#7076) secondary antibodies (Cell Signaling Technology). β-actin (A5316; Sigma-Aldrich) was used as a loading control. Immunopositive bands were visualized using WesternBright Sirius HRP substrate (Advansta, Menlo Park, CA, USA). Quantification of protein bands from three independent experiments was performed by densitometry using VisionWorks LS Acquisition and Analysis software (UVP, Upland, 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!