Plasmid Generation for Protein Aggregation

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).

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Button R.W., Roberts S.L., Willis T.L., Hanemann C.O, & Luo S. (2017). Accumulation of autophagosomes confers cytotoxicity. The Journal of Biological Chemistry, 292(33), 13599-13614.

Publication 2017

mRFP-GFP-LC3 Scramble shRNA mTOR shRNA

Cdna Ecori Mtor Plasmids Shrna α synuclein

Corresponding Organization : University of Plymouth

Top 5 similar protocols

Variable analysis

independent variables

MRFP-GFP-LC3 (catalog no. 21074)
HA-α-synuclein (catalog no. 40824)
GFP-α-synuclein A53T (catalog no. 40823)
HA-tagged HTT-72Q
GFP-HTT-21Q
GFP-HTT-72Q
MCherry-HTT-72Q
MCherry-α synuclein
Scramble shRNA (Addgene, 1864)
MTOR shRNA (Addgene, 1855)
STX-17 shRNA

dependent variables

Not explicitly mentioned

control variables

Not explicitly mentioned

positive controls

Scramble shRNA (Addgene, 1864)

negative controls

MTOR shRNA (Addgene, 1855)

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