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The Effects of BDNF on Nitric Oxide Synthase I Using Human SH-SY5Y Cells. By Amir Abyaneh, and Dr. Terrie Rife. Nitric Oxide Synthase I. Nitric Oxide Synthase I (NOS I) is an enzyme that catalyzes the reaction of a free radical, NO • . Learning and Memory Neurodegenerative Diseases/Problems

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The effects of bdnf on nitric oxide synthase i using human sh sy5y cells

The Effects of BDNF on Nitric Oxide Synthase I Using Human SH-SY5Y Cells

By Amir Abyaneh, and

Dr. Terrie Rife


Nitric oxide synthase i
Nitric Oxide Synthase I SH-SY5Y Cells

  • Nitric Oxide Synthase I (NOS I) is an enzyme that catalyzes the reaction of a free radical, NO•.

  • Learning and Memory

  • Neurodegenerative Diseases/Problems

  • Why do we study it?



Human nos i structure
Human NOS I Structure SH-SY5Y Cells

5’2

exon 2

exon 3- 29

5’4

5’3

5’1

{ >20 kB }

Transcription

Translation

Note - Figure is not drawn to scale


What we know
What We Know! SH-SY5Y Cells

Nerve Growth Factor

NEURON

p75

Trk A

NOS 1 ↑



Cell lines

p75 SH-SY5Y Cells

RA

Trk B

RA

RA

SH-SY5Y Cells

RA

RA

Cell Lines

  • human neuroblastoma cells SH-SY5Y

  • A model for neuronal differentiation

  • Responds to BDNF after being treated with Retinoic Acid 5 days


Establishing a protocol

Untreated SH-SY5Y Cells

Establishing a Protocol

RA w/ 10%FBS for 5 Days

BDNF for No Serum for 6 days


Methods
Methods SH-SY5Y Cells

  • Pretreatment with RA and 10% FBS for 5 Days

  • Treated with BDNF for 2, 4, 6, and 8 days, with a No Treatment control

  • RNA Harvesting

  • Reverse Transcription

  • PCR Analysis


The effects of bdnf on nitric oxide synthase i using human sh sy5y cells

Primers – PCR analysis SH-SY5Y Cells

RA

2

4

8

No DNA

NT

GADPH

380bp

nNOS

433bp


Future

5’2 SH-SY5Y Cells

exon 2

exon 3- 29

5’4

5’3

5’1

{ >20 kB }

Transcription

Translation

FUTURE

  • PCR analysis of transcripts:

  • 5’1

  • 5’2

  • 5’3

  • 5’4

Note - Figure is not drawn to scale


Thanks
Thanks SH-SY5Y Cells