Methods

1- to 3-days old male flies were homogenized by pestle, total RNA was extracted with REzole, and then 5 μg of the total RNA was reverse transcribed with random hexamer primer.

For each cDNA preparation, a control synthesis reaction was treated with DNase on 37°C for 25 mins to ensure that there was no contaminating genomic DNA. The resulting cDNA library was subjected to PCR with primers. The products of PCR were analyzed by electrophoresis on 1% agarose gels, and then visualized using ethidium bromide (EtBr).

rp49 gene was used as an internal standard among different lines.

2.2.2 Sample Preparation

Whole flies were washed 3 times with PBS, and then the total protein of the whole fly were extracted using ultrasonication with lysis buffer containing 8 M urea, 2% CHAPS, 1%

v/v Triton X-100, 0.1% w/v SDS, and 5 mM DTT. The extract was boiled at 95℃ for 5 minutes following centrifugation for 10 mins at 15000¯g for several times until the

10

supernatant was clear.

2.2.3 Protein Quantitation

Using BSA as a standard, protein quantitation of the homogeneous from D. melanogaster was estimated by a colorimetric assay (RC DC protein assay, Bio-Rad). Reagent A’ (2.5μl DC Reagent S and 125μl DC Reagent A) was prepared for each sample. Mix 25μl of sample (25X dilution with extraction buffer) with 125μl RC Reagent I. After gently vortex, 125μl of RC Reagent II was added, and centrifuge at 15000Xg for 10minutes. The protein pellet was mixed with 127μl Reagent A’, and then after the incubation for 5 minutes until the pellet was dissolved, added 1ml of DC Reagent B into the solution. Incubate the solution at room temperature for 15 minutes, and finally absorbance can be read at 750nm with UV/VIS spectrophotometer.

2.2.4 Two Dimensional Electrophoresis

IEF: 200μg or 500μg of the total proteins were loaded depending on the detection methods (silver/SYPRO Ruby stain or negative stain). The proteins were mixed with the rehydration buffer (8 M urea, 2% w/v CHAPS, 0.5% v/v IPG buffer pH 4-7, 0.002%

bromophenol blue, and 18.2 mM DTT), and then eletrofocused in DryStrip using the following protocol with 50μA per strip at 20℃: (1) rehydration for 12 hours; (2) 500V for 500VHr (step and hold); (3) 1000V for 1000VHr (step and hold); (4) 3000V for 3000VHr (gradient); (5) 5000V for 5000VHr (gradient); (6) 8000V for 8000VHr (gradient); (7) 8000V for 40000VHr (step and hold). For silver stain and SYPRO Ruby stain, the strips were shaken for 15 minutes with first equilibration buffer (6 M urea, 29.3% v/v glycerol, 2% w/v SDS, and 75 mM Tris-HCl at pH 8.8) which contain 1% DTT, and then were shaken for 15 minutes with second equilibration buffer which contain 2.5% IAA. For reverse stain, the strips were shaken for 2X15 minutes with equilibration buffer.

11

SDS-PAGE: the strips were transferred onto 12.5% second-dimensional SDS-PAGE (1.5mm for silver stain or 1.0mm for negative stain) with the following protocol: (1) 15mA/strip for 30minutes at 4℃; (2) 30mA/strip for 5.5 hours at 4℃.

2.2.5 Protein Staining and analysis of 2-DE gels

Silver stain: fixation (40% methanol, 10% acetic acid) for overnight; sensitization (30%

methanol, 0.2% w/v sodium thiosulphate, 0.5M sodium acetate) for 30 minutes; washing (ddH2O) for 3X5 minutes; silver reaction (0.25% w/v silver nitrate) for 20 minutes; washing (ddH2O) for 2X1 minute; developing (2.5% w/v sodium carbonate, 0.02% formaldehyde) for 3-5 minutes; stopping (0.05M EDTA) for 10 minutes; washing (ddH2O) for 3X5 minutes;

preserving (8.7% glycerol, 30% methanol) for 1 hour.

Reverse stain: fixation (40% methanol, 10% acetic acid) for overnight; neutralization (tris, glycine, SDS) for 2X30 minutes; solution I (200mM imidazole, 0.1% SDS) for 1 hour;

washing (ddH2O) for 2X 1 minute; solution II (300mM zinc sulfate) for 1-2 minutes; stopping (ddH2O).

SYPRO Ruby staining: fixation (40% methanol, 10% acetic acid) for overnight; the gel was staining with SYPRO Ruby protein gel stain (Bio-Rad) for 16-18 hours; Rinse the gel in 10% methanol and 7% acetic acid for 1 hour, which to decrease the background fluorescence;

finally was the gel before imaging. In SYPRO Ruby staining gel, it is readily visualized using a UV or blue light source box. 

Digital images of the gels were scanned by ImageScanner, and analyzed using ImageMaster 2D Platinum software V5.0 (GE Healthcare Bio-Science). The spots were detected and the background was subtracted (mode: average on boundary), and the gels were aligned and matched. A quantitative determination of the spots volumes was performed (mode: total spot volume normalization). Specific spots, either upregulated or downregulated, were excised for further identifying by MS analysis.

12

2.2.6 In-gel Digestion of Protein Spots in 2-DE gels

Specific spots, either upregulated or downregulated, were excised from the gels. The gel particles were washed with 50mM NH4HCO3/acetonitrile (1:1) for 15 minutes, and then silver destain solution (15mM K3Fe(CN)6, 50mM Na2S2O3) was used to destain the gel particles within 2X10 minutes. After removing the solution, we washed the gel particle in 20mM ammonium bicarbonate until it became colorless. The remaining liquid was removed, and then the gel particles were shrunken by adding just enough acetonitrile to cover the gel.

Finally, the gel particles were dried down for 15-20 minutes at room temperature. (In reverse stain, the gel particles were swelled in 10mM DTT/25mM NH4HCO3 for 1 hour at 56℃, and then we replaced the solution by 55mM IAA/25mM NH4HCO3 for 30minutes at room temperature. The gel particles were washed with 50mM NH4HCO3/acetonitrile (1:1) for 15 minutes, then shrunk and dried down following the steps described before.) The proteins in the gel particles were digested in the enzyme solution (25mM NH4HCO3 with 5ng/μl of trypsin) at 4℃ for 1 hour, and then incubated at 37℃ overnight by adding 3μl of 25mM NH4HCO3. The digests were sonicated in a water bath for 10 minutes, and then 50%

acetonitrile with 1% TFA was added. Finally, the supernatants were collected for analysis of MALDI-TOF-MS.

2.2.7 Protein Identification by MALI-TOF Mass Spectrometry

The MS raw data were processed by searching the protein databases (Swiss-Prot, MSDB, NCBInr) using MASCOT (http://www.matrixscience.com). To denote a protein as unambiguously identified, the Mowse scoring algorithms were used. Only proteins whose score exceeded the significance threshold (P<0.05) were concerned.

2.2.8 Oxidation Stress Assay of Flies from APPL-GAL4>UAS-TPST

, APPL-GAL4/+, and UAS-TPST

/+

13

The young male flies about 1- to 3-days old were collected for oxidative stress test.

The flies were fed with 10mM paraquat in 5% sucrose water once a day (16:30). The dead fly number was counted every 4 hours (00:30, 8:30, 12:30, 16:30, 20:30) till all flies were dead. Each tube contained about 20 flies, and at least 100 flies were included for each line.

The statistical significance of the observed change in the stress test was evaluated by using Student’s t test.

14