*4.7. Western Blot Analysis*

Cells were lysed in lysis solution (Ambion, Grand Island, NY, USA) and incubated at 95 ◦C for 10 min. Protein concentration was determined by the Bradford assay kit (Takara Biotechnology, Dalian, China). Twenty micrograms of total proteins was separated by 10%–12% sodium dodecyl sulfate polyacrylamide gels and then transferred to polyvinylidene difluoride membranes. Blots were probed with rabbit monoclonal anti-Akt (1:1000), anti-pAkt (1:1000), anti-PI3K (1:1000), anti-pPI3K (1:1000). Blots were also probed with rabbit monoclonal anti-GAPDH antibody (Milwaukee, WI, USA, Sigma, 1:10,000) as a loading control. Anti-rabbit secondary antibodies conjugated to horseradish peroxidase were used at 1:10,000 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). UVP BioSpectrum ®CCD imaging system (Davis, CA, USA) was used for imaging and analysis. Camera settings were manipulated in preview mode to optimize the exposure and determine the appropriate final exposure settings. Exposures of 30 s up to 5 min were used for data collection. Results were analyzed through scanning densitometry by UVP Vision Works LS Software (UVP, Cambridge, UK).

#### *4.8. Total RNA Extraction and Real Time PCR*

SH-SY5Y Cells were seeded in 6-wells plates and incubation with MPP<sup>+</sup> and di fferent reagen<sup>t</sup> for 24 h. Total RNA was isolated by Trizol Reagent (Takara Biotechnology, Dalian, China) ccording to the manufacturer's instructions. From each sample, 1 μg of total RNA was retrotranscripted into cDNA (Takara RR047A, Dalian, China). Then, 2 μL of each sample was used as a template for amplification reactions conducted with the SYBR Premix Ex TaqTMII (Takara Biotechnology, Dalian, China) following the manufacturer's instructions. The PCR amplifications were conducted using a life Technology 7500 fast Real-time PCR system. The expression of house-keeping gene, GAPDH mRNA, was served as the standardized control. Primer (showed in Table 2) selection was performed using the Primer Premier Design Software, version 1.0 (Idaho Technology, Inc., Alameda, CA, USA). The mRNA level for the control group was set as 100%.


#### **Table 2.** Primers used for real-time RT-PCR.

## *4.9. Caspase-3, -8 and -9 Activity*

After treatment of cells with UF for 24 h, the cells were harvested using cell scrapers and washed in ice-cold PBS. Then, the cells were lysed for 30 min on the ice in 100 μL of Cell Lysis Reagent supplemented with complete protease inhibitor cocktail. The protein concentration of cell lysates was determined by Bicinchoninic acid (BCA) assay (Takara Biotechnology, Dalian, China).
