Neuron Culture and Antibody Protocol

Mouse α-βIII tubulin antibody was prepared in house (20 (link)), Rabbit anti- βIII (T2200) was purchased from Sigma Aldrich, Alexa Fluor 488 cross-linked Goat anti-Mouse (A11029) and anti-Rabbit (A11034) antibodies were purchased from Life Technologies. Primary antibodies α-GSK3β (9832), α-cJun (3270), α-Cofilin-1 (5175), α-Erk1/2 (9107), α-NF-κB (6956), α-pSer9 GSK3β (5558), α-pSer73 c-Jun (2315), α-pThr202/Tyr204 Erk1/2 (4370), and α-pSer276 NF-κB (3031) were purchased from Cell Signaling; α-pSer3 Cofilin-1 (sc-271923) was purchased from Santa Cruz Biotechnology. Secondary antibodies α-mouse-[IR800] (926-32210) and α-mouse-[IR680] (926-32220) were purchased from LiCor, while α-rabbit-[IR800] (611-732-127) and α-rabbit-[IR680] (611-130-002) were purchased from Rockland. Poly-D-Lysine (P7886-500MG) and sterile dimethyl sulfoxide (DMSO) (D2650) were purchased from Sigma Aldrich. Hippocampal tissue was incubated in Hibernate E from BrainBits, supplemented with NeuroCult® SM1 (05711) from StemCell Technologies. Neurons were cultured in NbActive4® media from BrainBits. Accell siRNA SMARTpools were purchased from Thermo Scientific. All other reagents were purchased from Life Technologies. In vitro kinase profiling was done at NanoSyn.

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Al-Ali H., Lee D.H., Danzi M.C., Nassif H., Gautam P., Wennerberg K., Zuercher B., Drewry D.H., Lee J.K., Lemmon V.P, & Bixby J.L. (2015). Rational polypharmacology: systematically identifying and engaging multiple drug targets to promote axon growth. ACS chemical biology, 10(8), 1939-1951.

Publication 2015

Hibernate E NeuroCult® SM1 NbActive4® media

Alexa fluor 488 Antibodies Antibody Cofilin 1 Cultured media Dimethyl sulfoxide Erk1 Goat Gsk3β Kinase Lysine Mouse Neurons Nf κb Poly Rabbit Sirna Stemcell Sterile Tissue Tubulin

Corresponding Organization :

Other organizations : Amazon (United States), Institute for Molecular Medicine Finland, Finland University, University of Helsinki, Research Triangle Park Foundation, GlaxoSmithKline (United States)

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Variable analysis

independent variables

Mouse α-βIII tubulin antibody prepared in house
Rabbit anti- βIII (T2200) purchased from Sigma Aldrich
Alexa Fluor 488 cross-linked Goat anti-Mouse (A11029) and anti-Rabbit (A11034) antibodies purchased from Life Technologies
Primary antibodies α-GSK3β (9832), α-cJun (3270), α-Cofilin-1 (5175), α-Erk1/2 (9107), α-NF-κB (6956), α-pSer9 GSK3β (5558), α-pSer73 c-Jun (2315), α-pThr202/Tyr204 Erk1/2 (4370), and α-pSer276 NF-κB (3031) purchased from Cell Signaling
α-pSer3 Cofilin-1 (sc-271923) purchased from Santa Cruz Biotechnology
Secondary antibodies α-mouse-[IR800] (926-32210) and α-mouse-[IR680] (926-32220) purchased from LiCor, while α-rabbit-[IR800] (611-732-127) and α-rabbit-[IR680] (611-130-002) purchased from Rockland
Poly-D-Lysine (P7886-500MG) and sterile dimethyl sulfoxide (DMSO) (D2650) purchased from Sigma Aldrich
Hippocampal tissue incubated in Hibernate E from BrainBits, supplemented with NeuroCult® SM1 (05711) from StemCell Technologies
Neurons cultured in NbActive4® media from BrainBits
Accell siRNA SMARTpools purchased from Thermo Scientific

dependent variables

Not explicitly mentioned

control variables

Poly-D-Lysine (P7886-500MG) and sterile dimethyl sulfoxide (DMSO) (D2650) purchased from Sigma Aldrich

controls

Positive control: Not specified
Negative control: Not specified

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