Supplementary Methods s4

Supplementary methods

Animals. Male Sprague-Dawley rats (300–350 g body weight) and male C57BL/6J mice (25-35 g body weight) were purchased from Tagawa experimental animals. Animals were maintained on a 12-h light/dark cycle at a room temperature of 23 ºC with free access to food and water. Animal care and experimental procedures were performed in accordance with conformed to the Guidelines for Animal Experimentation of Nagasaki University with approval of the Institutional Animal Care and Use Committee.

Materials. Antibodies used were purchased from Santa Cruz Biotechnology (CA, USA) for BDNF, EPO and Myc, and from Cell signaling (Tokyo, Japan) for activated-caspase 3. Propidium iodide (PI) and triphenyltetrazolium chloride (TTC) were purchased from Sigma (Tokyo, Japan).

Preparation of recombinant proteins. Purifications using acidic phenol of recombinant rat and mouse ProT (rrProT and rmProT) were performed as described previously.1 In the recombinant protein preparation, ProT gene was first amplified from cDNA derived from rat or mouse brain using specific primers (rat and mouse 5’ primer, 5’- AACATATGTCAGACGCGGCAGTGGA-3’; rat 3’-primer, 5’- AAGGATCCAGTGGAGGGTGAATAGGTCAC-3’), mouse 3’-primer, 5’- GGATCCAAGCTTGCTGTCTAGTCATCCTGG-3’), respectively. The PCR product was cloned into pGEM-T Easy, and then subcloned into pET16b. BL21 (DE3) cells were transformed with pET16b-ProT. rrProT or rmProT expression was induced by 0.1 mM isopropylthio--galactopyranoside. Expressed rrProT or rmProT was purified by acidic phenol method, applied to an ion exchange-membrane spin column (Vivapure Q MiniTM; Sartorius KK), which had been equilibrated with 20 mM sodium acetate, pH 5.2. The sample was eluted with different concentrations of NaCl (0.2-1 M), and then dialyzed against phosphate-buffered saline (PBS) for later use. They were then used for experiments using rat or mouse preparations, respectively. Brain ischemia-reperfusion stress. Middle cerebral artery occlusion (MCAO) was performed, according to a conventional procedure. Male Sprague-Dawley rats were anesthetized by isoflurane mixture during the surgical procedure. MCAO was carried out by use of monofilament nylon surgical suture (3-0 in size). The monofilament was removed 60 min after occlusion to restore the blood flow. Animals were sacrificed 24 h later for neuropathological analysis unless otherwise stated. Unfixed 1 mm coronal brain slice was incubated with 2 % TTC in PBS (pH 7.4). Brain infarct volume was determined using Swanson correction.2 Clinical score was evaluated, as elsewhere reported.3, 4 Bilateral common carotid artery occlusion (BCCAO) using male C57BL/6J mice (25–35 g body weight, from Tagawa Experimental Animals, Nagasaki, Japan) was performed, as previously reported.5 Under anesthesia, common carotid arteries were exposed through a midline incision in the neck and ligated with 5-0 silk threads. Body temperature was maintained at 37.0 °C with heating plate at the time of occlusion. For morphological analysis, the brain pretreated with BCCAO was carefully removed, fixed with 4% paraformaldehyde (PFA) and embedded in paraffin. Thin sections (5 mm thick) were then cut and stained with hematoxylin and eosin. Learning and memory deficits were evaluated with step-through passive avoidance test.5

Immunostaining of brain sections. In the in vivo PI-staining, treated brain slice (1 mm) was incubated with 10 g/ml PI in DMEM at 25°C for 30 min, washed three times with DMEM, followed by cutting cryosections (30 m), fixation using 4% PFA and cytoprotection in 30% sucrose at 4 °C overnight. The sections were then rinsed twice with PBS and pre-incubated in blocking buffer (2% BSA with 0.1% Tween 20 in PBS) for 1 h at 25°C. Next, the sections were incubated with activated caspase-3 antibodies in blocking buffer overnight at 4°C, rinsed with PBS and incubated with FITC-conjugated anti-rabbit IgG (1:200; Santa Cruz Biotechnology) for 2 h at 25°C. The immunolabeled sections were mounted with Permafluor (Thermo Shandon, Pittsburgh, PA). For imaging cells, a laser scanning confocal microscope imaging system (Microscope: Axiovert 200 M, Scan module: LSM 5 PASCAL; Carl Zeiss MicroImaging, Inc.) with image browser software (Carl Zeiss MicroImaging, Inc.) were used at ambient temperature, equipped with 40x/1.3 oil-immersion lens and non-imaging photodetection device (photomultiplier tube; Carl Zeiss MicroImaging, Inc.). Immersion oil (Immersol 518; Carl Zeiss MicroImaging, Inc.) was used for imaging medium. A dynamic range adjustment was used to optimize the signal for the fluorophores, and images were collected in multi-track mode (Carl Zeiss MicroImaging, Inc.). Brightness and contrast adjustments were performed in Photoshop (Adobe).

ProT determinations. Brain Myc-tagged ProT transport following systemic injection (1 mg/kg i.p.) was evaluated by acidic Western blot analysis using anti-Myc IgG. Brain lysates (30 g/lane) were subjected to 15% SDS-PAGE and electrotransferred onto nitrocellulose membrane in 20 mM sodium acetate buffer, pH 5.2, followed by fixation with 0.5% glutaraldehyde (acidic blotting)6, 7 and probing with polyclonal anti-Myc IgG. Immunoreactive bands were visualized using an enhanced chemiluminescent substrate (Super Signaling Substrate; Pierce Chemical Co., Rockford, IL) for the detection of horseradish peroxidase.

Statistical Analysis. Multiple comparisons of analysis of variance (ANOVA) followed by Student’s t-test were used for statistical analysis of the data. The criterion of significance was set at P < 0.05. All results are expressed as the mean ± S.E.M. References 1 Evstafieva AG et al. Eur J Biochem 1995; 231: 639-643. 2 Shibata M et al. Circulation 2001; 103: 1799-1805. 3 Kagiyama T et al. Stroke 2004; 35: 1192-1196. 4 Longa EZ Weinstein PR Carlson S Cummins R Stroke 1989; 20: 84-91. 5 Nakao K et al. Cell Mol Neurobiol 1998; 18: 429-436. 6 Kondili K Tsolas O Papamarcaki T Eur J Biochem 1996; 242: 67-74. 7 Sukhacheva EA et al. J Immunol Methods 2002; 266: 185-196.