MOVAS cell line (mouse aortic root vascular smooth muscle cell line) was obtained from ATCC (MD, USA) and cultured in DMEM (GIBCO, LA, USA) containing 10% fetal bovine serum (Sigma, CA, USA). The cells were transfected with constructs p3×Flag-CMV-Ccng2 or empty plasmid p3×Flag-CMV-14. These were labeled as experimental group (FLAG-Ccng2) and control group (FLAG-Vector) correspondingly. In MOVAS cells, foaming was induced using 80 µg/mL ox-LDL (Yi-yuan, Guangzhou, China) for 24 hours. The PP2A activator DT-061 (MedChemExpress, CA, USA), at a dose of 20 µM or equal volume of DMSO (Sigma, CA, USA), was added to respective groups and these were denoted as FLAG-Vector + DMSO, FLAG-Ccng2 + DMSO, FLAG-Ccng2 + DT-061, and FLAG-Vector + DT-061 groups respectively.
Dt 061
Manufactured by Merck Group
Sourced in United States
The DT-061 is a laboratory equipment manufactured by Merck Group. It is designed for general purpose laboratory applications. The core function of the DT-061 is to perform precise measurements and analysis of samples.
Automatically generated - may contain errors
Lab products found in correlation
Puromycin, by Merck Group (1 mentions)
Dt 061, by MedChemExpress (1 mentions)
Movas cell line, by American Type Culture Collection (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Camkkβ, by Abcam (1 mentions)
P ampk, by Abcam (1 mentions)
Compound c, by Merck Group (1 mentions)
Gapdh, by Abcam (1 mentions)
Okadaic acid, by Abcam (1 mentions)
Ar a014418, by Cayman Chemical (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
4 protocols using dt 061
1
Ccng2 Overexpression in Ox-LDL-Induced MOVAS Cells
2
Measuring Nascent Protein Translation
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Nascent protein translation was measured utilizing the SUrface SEnsing of Translation (SUnSET), a radioactive free puromycin-based method previously described by Schmidt et al.72 (link). Pulse-chase experiments were performed as follows: Cells were plated overnight at 37°C in 5% CO2. The next day, cells were then treated with either DMSO or DT-061. One hour prior to harvesting, 10 minutes of 10μg/ml puromycin (Sigma-Aldrich, P8833) incubation (pulse) was performed to label nascent polypeptide chains, followed by 50-minute incubation with puromycin-free media containing either DMSO or DT-061 (chase). One wash of cold 1X Phosphate Buffer Saline (PBS) (Fisher Scientific, SH3025601) was used in between the two phases. Cells were then harvested and puromycin labeling was measured via western blotting techniques.
3
Lipid Metabolism Regulation Assays
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Triglyceride (TG), total cholesterol (TC), and low-density lipoprotein-cholesterol (LDL-c) were from Roche Life Science (Basel, Switzerland). 2-Deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG), STO-609 (CaMKKβ inhibitor), compound C (AMPK inhibitor), and DT-061 (PP2A activator) were from Sigma-Aldrich (St. Louis, United States), and PP2A, AMPK, p-AMPK, CaMKKβ, p- CaMKKβ, and GAPDH were purchased from Abcam (Cambridge, UK).
4
Differentiation of Osteoclasts from Bone Marrow Macrophages
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To prepare BMMs and osteoclasts, we followed the previously described protocol [20 (link)] using wild-type (Pcdh7+/+) and Pcdh7-deficient (Pcdh7−/−) mice. The femurs and tibias were extracted, and the whole bone marrow was incubated in α−MEM (Invitrogen, Carlsbad, CA, USA) medium containing 10% fetal bovine serum and M−CSF (5 ng/mL) overnight in 100 mm petri dishes. Non-adherent cells were collected, and BMMs were generated by culturing the cells with M−CSF (60 ng/mL) for three days. To prepare preosteoclasts, BMMs were cultured with M−CSF (60 ng/mL) and RANKL (150 ng/mL) for one day. For osteoclast differentiation, BMMs were cultured with M−CSF (60 ng/mL) and RANKL (150 ng/mL) for three days. Osteoclasts were stained using Leukocyte acid phosphatase Lit (387A−1KT, Sigma, St. Louis, MO, USA) according to the manufacturer’s instructions. Okadaic acid (Cat.# ab120375) was purchased from abcam (Cambridge, MA, USA); Cytostatin (Cat.# 19602), LB−100 (Cat.# 29105), and Rubratoxin A (Cat.#19605) were purchased from Cayman chemical (Ann Arbor, MI, USA); AR−A014418 (Cat.# A3230) was purchased from Sigma (St. Louis, MO, USA); and DT−061 (Cat.# S8774) was purchased from Shelleckchem, Houston, TX, USA.
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