Plasmids mRFP-GFP-LC3 (catalog no. 21074) (28 (link)), HA-α-synuclein (catalog no. 40824), and GFP-α-synuclein A53T (catalog no. 40823) (59 (link)) were obtained from Addgene. HA-tagged HTT-72Q (60 (link)), GFP-HTT-21Q, and GFP-HTT-72Q (61 (link)) were described previously. To obtain mCherry-HTT-72Q and mCherry-α synuclein, mCherry cDNA was cloned into pcDNA3 flanked with HindIII/BamHI sites. To ligate HTT-72Q into pcDNA mCherry, HTT-72Q was cut with BglII/EcoRI, and pcDNA mCherry was cut with BamHI/EcoRI. To ligate α-synuclein A53T into pcDNA mCherry, α-synuclein was cut with BamHI/XhoI, and pcDNA3 mCherry was cut with BglII/SalI. Plasmids were confirmed by DNA sequencing. Scramble shRNA (Addgene, 1864) and mTOR shRNA (Addgene, 1855) was a gift from Dr. David Sabatini (62 (link)). STX-17 shRNA was purchased from Sigma (TRCN0000379933).
Mtor shrna
Manufactured by Addgene
The mTOR shRNA is a short hairpin RNA (shRNA) targeting the mTOR gene. It is designed to knockdown the expression of the mTOR (mechanistic target of rapamycin) protein in cells. The mTOR protein is a serine/threonine protein kinase that regulates cell growth, proliferation, and metabolism.
Automatically generated - may contain errors
2 protocols using mtor shrna
1
Plasmid Generation for Protein Aggregation
2
Modulating MSC Differentiation Pathways
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BMMSCs (0.5 × 106) were seeded to a 6-well culture plate and treated with Fbn1 siRNA (Santa Cruz Biotechnology, Inc.), P70s6k siRNA (Santa Cruz Biotechnology, Inc.), Il4rα shRNA (Santa Cruz Biotechnology, Inc.), or Mtor shRNA (Addgene), according to the manufacturers’ instructions. After transfection, cells were either used for protein extraction for Western immunoblotting or for differentiation induction. For chemical reagent treatments, serum-starved MSCs were treated with 10–100 ng/ml rIL-4 (R&D Systems), 50 nM rapamycin (LC Laboratories), or 1 µg/ml TGF-β neutralized antibody (R&D Systems). For Western immunoblotting, MSCs were cultured under growth medium with drugs for 24 h, and protein was extracted by using M-PER mammalian protein extraction reagent. For differentiation induction, MSCs were cultured under inductive conditions in the presence of drugs (added every 3 d) for 3 wk, followed by staining and gene expression analysis.
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