SEARCH

SEARCH BY CITATION

Supplementary methods Cloning of wild type or mutant A30P, A53T a-syn genes into pEGP-C1 plasmids.

Fig. S1 Overexpression of human WT, A30P and A53T α-syn in dopaminergic PC12 cells after transient transfection with pEGFP-C1 plasmids cloned with or without human WT or mutant α-syn genes. (a–d) PC12 cells were cultured for 72 h after transfection with pEGFP-C1 plasmids cloned with or without human WT, A30P, and A53T α-syn genes and observed under fluorescent microscopy. (a) EGFP plasmid; (b) EGFP-WT plasmid; (c) EGFP-A30P plasmid, and (d) EGFP-A53T plasmid. (e) Verification of pEGFP-C1 plasmids cloned with or without human WT, A30P, and A53T α-syn genes by PCR and BigII and SalI double restriction endonuclease digestion. M is for 100 bp DNA marker; lane 1 is human WT α-syn fragment after PCR; lane 2 is WT α-syn fragment after further PCR with adapter primers; lane 3 is intact EGFP plasmid; lane 4 is EGFP plasmid after endonuclease digestion; lane 5 is intact EGFP-WT plasmid; lane 6 is EGFP-WT plasmid after digestion; lane 7 is intact EGFP-A30P plasmid; lane 8 is EGFP-A30P plasmid after digestion; lane 9 is intact EGFP-A53T plasmid; and lane 10 is EGFP-A53T plasmid after digestion.

Fig. S2 Transient transfection and overexpression of human wild type and mutant A30P, A53T α-syn in dopaminergic MN9D and non-dopaminergic N2A cells.  (a–h), MN9D and N2A cells were transfected with pEGFP-C1 plasmids cloned with or without human WT, A30P, and A53T α-syn genes and observed under fluorescent microscopy after 72 h of transfection. (a) MN9D with EGFP plasmid; (b) MN9D with EGFP-WT plasmid; (c) MN9D with EGFP-A30P plasmid; (d) MN9D with EGFP-A53T plasmid; (e) N2A with EGFP plasmid; (f) N2A with EGFP-WT plasmid; (g) N2A with EGFP-A30P plasmid, and (h) N2A with EGFP-A53T plasmid. (i and j), Overexpression of human wild type and mutant α-syn in MN9D and N2A cells, detected by western blot analysis. MN9D (i) and N2A (j) cells were harvested after transfection with various EGFP plasmids for 72 h. Cell lysates were analyzed by western blot analysis with antibodies against GFP or α-syn respectively. Lane 1, without transfection; lane 2, transfected with pEGFP-C1 plasmid; lane 3, with EGFP-WT plasmid; lane 4, with EGFP-A30P plasmid, and lane 5, with EGFP-A53T plasmid.

Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

FilenameFormatSizeDescription
JNC_5795_sm_Fig S1.jpg239KSupporting info item
JNC_5795_sm_Fig S2.jpg334KSupporting info item
JNC_5795_sm_Supplementary methods.doc24KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.