Semi-quantitative western analysis of VEGF-B expression. Midbrain cultures were exposed to 3 concentration of rotenone (16 nM, 25 nM and 40 nM) for 5 days. Protein was isolated and a western analysis was performed using a procedure previously published . We used the expression of the house keeping β-actin protein (anti-β-actin antibody from Sigma-Aldrich, St. Louis, MO) as the internal standard for the relative quantification of VEGF-B protein levels (anti-VEGF-B antibody from Santa Cruz Biotechnology, Santa Cruz, CA). A concentration-dependent increase in VEGF-B protein level was evident. In (A) example western blots for VEGF-B (on the left) and β-actin (on the right) are shown; the samples were run in triplicate. This was repeated with 3 separate preparations. The summary graph in (B) depicts the mean relative VEGF-B level (± s.e.m.) that was determined by densitometry using Image-J (Wayne Rasband, Bethesda, MD). Statistical significant differences from control were determined by one-way ANOVA followed by a Fisher LSD post hoc test, and are depicted by asterisks (*P < 0.05, **P < 0.001). 40 nM rotenone induced a significantly greater increase than 16 nM and 25 nM rotenone (*P < 0.05). The statistical analyses were conducted using SPSS software, version 16.0 for Windows (SPSS, Chicago, IL).