a. mAng1 is the most downregulated gene in a gene microarray study evaluating gene expression in the substantia nigra of transgenic α-syn mice compared to wildtype littermates. The top 20 most downregulated genes are shown from three-month-old mice. This data was extracted from the supplemental data of our previous publication (Yacoubian et al. 2008).
b. mAng1 protein levels are also reduced in the cortex of three-month-old transgenic mice. Representative Western blot from lysates from transgenic and wildtype mouse cortices.
c. Quantification of Western blotting against angiogenin by densitometry. Tubulin was used as a loading control, and angiogenin levels were normalized to tubulin. **p<0.01 (unpaired t-test). Error bars reflect SEM.
d. Angiogenin expression is not altered in the cortex of three-month-old α-syn knockout mice. Representative Western blot from lysates of knockout and wildtype mouse cortex homogenate.
e. Quantification of Western blotting against angiogenin by densitometry. Tubulin was used as a loading control, and angiogenin levels were normalized to tubulin. ns = no significant difference (unpaired t-test). Error bars reflect SEM.

a. Representative Western blot demonstrates increased Akt phosphorylation at serine 473 within 30 seconds following treatment of SH-SY5Y cells with recombinant angiogenin (100nM). This Akt phosphorylation is maintained for several hours. Total Akt levels are unchanged with angiogenin treatment. Actin is used as a loading control.
b. Densitometric quantification of Western blots against phosphorylated Akt. Results reflect three independent experiments. **p<0.01 (One-way ANOVA with Tukey’s post-hoc test). Error bars reflect SEM.

a. Representative Western blot against cleaved caspase-3 of SH-SY5Y cells treated with 1mM MPP+ with or without 100nM angiogenin. Tubulin was used as a loading control.
b. Densitometric quantification of Western blots against cleaved caspase-3. Results reflect three independent experiments. Each condition was normalized to MPP+-treated condition for the given blot. **p<0.01 (one group t-test with Bonferroni correction). Error bars reflect SEM.

a. Western blotting reveals endogenous levels of angiogenin produced by both M17 and SH-SY5Y dopaminergic cell lines. Tubulin was used as loading control.
b. A robust increase in intracellular angiogenin staining is observed after treatment with exogenous angiogenin. SH-SY5Y cells were immunostained using a goat polyclonal antibody against angiogenin and a cy3-conjugated donkey anti-goat antibody at A) control, B) 2 hours, C) 12 hours, and D) 36 hours after treatment with 100nM recombinant angiogenin. Scale bar represents 25μm.

a. Angiogenin demonstrates a concentration-dependent protective response to MPP+ induced cell death. Cells were plated 150,000 cells/well and pretreated with varying concentrations of angiogenin for 12 hours prior to treatment with 0.5mM MPP+. Cell death was assayed by LDH release into media. n=6. ***p<0.001 (one-way ANOVA followed by post-hoc Bonferroni’s test). Error bars reflect SEM.
b. Angiogenin’s neuroprotective effect against MPP+ varies with cell density. SH-SY5Y cells were plated at cell densities ranging from 30,000 cells/well up to 125,000 cells/well and pretreated with 100nM angiogenin for 12 hours prior to treatment with 1 mM MPP+. Cell death was assayed by LDH release. n=4. ***p<0.001 (one-way ANOVA followed by post-hoc Bonferroni’s test). Error bars reflect SEM.

a. Exogenous angiogenin reduces rotenone-induced toxicity in SH-SY5Y cells in a cell-density-dependent manner. Cells were plated at either 30,000 or 50,000 cells/well and pretreated with angiogenin (100nM) prior to treatment with 0.2μM rotenone. n=4.
b. Angiogenin reduces rotenone toxicity in a different dopaminergic cell line, M17. n=4. *p<0.05 (one-way ANOVA followed by post-hoc Tukey’s test). Error bars reflect SEM