Research
Cathepsin D expression level affects alpha-synuclein processing, aggregation, and toxicity in vivo
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* Corresponding authors: Michael G Schlossmacher mschlossmacher@ohri.ca - Jaana Tyynelä jaana.tyynela@helsinki.fi
1 Center for Neurologic Diseases, Department of Neurology, Brigham & Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA
2 Institute of Biomedicine/Biochemistry, Helsinki University, Helsinki, Finland
3 Folkhälsan Institute of Genetics, Biomedicum Helsinki, Helsinki, Finland
4 Department of Pathology, Haartman Institute, Helsinki University, Helsinki, Finland
5 Department of Pathology, Brigham & Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA
6 Biochemical Institute, University of Kiel, Kiel, Germany
7 Division of Neuroscience, Ottawa Health Research Institute, Ottawa, Canada
8 Department of Pathology, University of Ottawa, Ottawa, Canada
9 Current affiliation : Link Medicine Corp., Cambridge, Massachusetts, USA
Molecular Brain 2009, 2:5 doi:10.1186/1756-6606-2-5
Published: 9 February 2009Additional files
Additional file 1:
Supplementary Figure 1 – Expression and quantification of α-synuclein in MES23.5 cells. Recombinant, human α-synuclein (aSyn) (A) and lysates of MES23.5 cells expressing αS (B-C) were analyzed by ELISA [hSA2/Biotinylated Syn-1]. (A) A typical standard curve of serial dilutions of recombinant αS (mean of triplicate wells). (B) Following generation of MES-aSyn cells with three different concentrations of human SNCA cDNA (μg/10 cm dish; total DNA transfected per dish, 5.5 μg), lysates were analyzed in 4 different dilutions, as indicated. Results show the mean absorbance signals of triplicates from a representative experiment. (C) Linear relationship between amount of SNCA-encoding cDNA transfected into MES23.5 cells and αS protein expression, as monitored by ELISA. Each lysate was analyzed in 4 different dilutions in triplicate. Linear regression of cDNA-to-protein was performed; results represent the mean aSyn values (μg) expressed from a representative experiment.
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Additional file 2:
Supplementary Figure 2 – Characterization of affinity-purified antibodies to α-synuclein using genotyped mouse brain. Whole brain extracts of genotyped mice were generated by lysis buffer that contained NP-40 and protease inhibitors [37]; increasing amounts of the NP-40 extract (μg/lane) were loaded onto SDS/PAGE gels under reducing conditions. Immunoblots were probed with polyclonal, affinity-purified anti-aSyn, hSA-2 (top panel) and mSA-1 (third panel). Loading controls showing IgG heavy chains are shown for both blots. Lysates were prepared from from snca knock-out mice without a transgene (no transgene; left half) and mice that carry a human, wild-type SNCA transgene (SNCA transgene; right half; brains kindly provided by Dr. Bob Nussbaum, UCSF). Note the specific detection of full-length aSyn (16 kDa), and of 12.5 kDa and ~10 kDa truncated species of aSyn in SNCA-expressing mice.
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