Plasmid Construction for TDP43 Proteins

The following constructs have been described. Human TDP43 cDNA (MGC #3506121; Thermo Fisher Scientific, Waltham, MA) was fused to meGFP45 (link), mKate2 (Evrogen, Moscow, Russia), mTFP1 (Allele Biotechnology, San Diego, CA), and mVenus46 (gift from Prof. T. Nagai); TDP43-GFP, R-TDP43-G, T-TDP43-Y, respectively. The plasmids harboring EGFP-TDP35 and EGFP-TDP25 (gifts from Dr. Y. J. Zhang and Prof. L. Petrucelli) sequence subcloned into the meGFP-C1 vector29 (link) (GFP-TDP35 and GFP-TDP25, respectively). NLS sequences were incorporated into GFP-TDP25 between GFP and TDP25 sequence (GFP-NLS-TDP25 or GFP-NLS^NP-TDP25). FLAG-tagged TDP43 and the CTFs were modified from GFP-tagged plasmids (FLAG-TDP43/35/25/NLS-TDP25). pBOS-H2B-mSECFP (H2B-CFP) and pBOS-H2B-iRFP (H2B-iRFP) was created using pBOS-H2B-GFP47 (link), pRSETb-mSECFP (gift from Prof. T. Nagai), and piRFP (Addgene plasmid #31857). LSSmOrange-DEVD-mKate2 (Addgene plasmid #37132) was used as a sensor for caspase 3-activation. Human ubiquitin sequence (IMAGE #5766897, Thermo Fisher Scientific) subcloned into mCherry-C1 (RFP-Ub). Detailed construction procedures were described in Supplemental Information.

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Kitamura A., Nakayama Y., Shibasaki A., Taki A., Yuno S., Takeda K., Yahara M., Tanabe N, & Kinjo M. (2016). Interaction of RNA with a C-terminal fragment of the amyotrophic lateral sclerosis-associated TDP43 reduces cytotoxicity. Scientific Reports, 6, 19230.

Publication 2016

mKate2 piRFP

Allele Caspase 3 Cdna Gifts Human Human ubiquitin Plasmid Tdp43

Corresponding Organization :

Other organizations : Hokkaido University

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Variable analysis

independent variables

Human TDP43 cDNA
MeGFP45
MKate2
MTFP1
MVenus46
EGFP-TDP35
EGFP-TDP25
GFP-NLS-TDP25
GFP-NLS^NP^-TDP25
FLAG-TDP43/35/25/NLS-TDP25
PBOS-H2B-mSECFP (H2B-CFP)
PBOS-H2B-iRFP (H2B-iRFP)
LSSmOrange-DEVD-mKate2
MCherry-C1 (RFP-Ub)

dependent variables

Not explicitly mentioned

control variables

Not explicitly mentioned

positive controls

Not specified

negative controls

Not specified

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