Uvichemi

CD8⁺ T cells were lysed with RIPA buffer (1 mM phenylmethylsulphonylfluoride [PMSF], 5 mM EDTA, 1 mM sodium orthovanadate, 150 mM sodium chloride, 8 μg/ml leupeptin, 1.5 % Nonidet P-40, 20 mM tris–HCl, pH 7.4) and 40 μg of each lysate were separated by SDS-PAGE and electroblotted onto a nitrocellulose filter. The filter was blocked overnight at RT with 10 % BSA in PBS containing 0.1 % Tween-20 (TBS) and incubated with anti-human JAK3 and anti-human MCL-1 Abs in 5 % BSA-TBS (clones B-12 and B-6 respectively; 1:250 dilution, Santa Cruz Biotechnology) overnight, at 4 °C. The membranes were washed in TBS-T 0.1 % and incubated for 1 h at RT with horseradish-peroxidase-conjugated sheep anti-mouse IgG at 1:5000 dilution in TBS-T 0.1 %. The membrane was then visualized using the ECL enhanced chemiluminescence system (Amersham, Little Chalfont, UK). The same membranes were then immunoblotted again with anti-actin antibody (clone MM2/193; 1:2000 dilution; Santa Cruz Biotechnology). Signal detection and densitometric analysis of the bands was performed using a UVIchemi (UVItec, Cambridge) digital imaging system and UVI1D software (UVItec, Cambridge).

Brains tissues from sham-operated control and pMCAO (3 to72 hr) rats were lysed by RIPA buffer (Cell Signaling) supplemented with protease inhibitor and phosphatase inhibitor cocktail (Roche, Mannheim, Germany). SH-SY5Y cells and primary cortical astrocytes were lysed by CST buffer (Cell Signaling) supplemented with protease inhibitor and phosphatase inhibitor cocktail (Roche, Manheim, Germany). Total protein was determined by Bradford protein assay (Biorad, CA, USA). Proteins were separated by 10% SDS/PAGE, transferred onto a polyvinylidene difluoride (PVDF) membrane (Amersham Biosciences, Buckinghamshire, UK), and then blocked with 10% nonfat milk. The membrane was then incubated with antibodies against CBS, or β-actin (Cell Signaling Technologies, Beverly, MA, USA) at 4 °C overnight, then washed and incubated in HRP-conjugated anti-rabbit or mouse IgG at room temperature for 1 hr. Visualization was carried out using Luminata Forte or Crescendo western HRP substrate (Millipore Corporation, Billercia, MA, USA), and the chemiluminescence signals were detected using UVIchemi (UVItec, Cambridge, UK).

The cortex was dissected from control and embolic stroke rats according to Spijker [27 ]. Cortical tissues were lysed by RIPA buffer (Cell Signaling Technologies, USA) supplemented with a protease inhibitor cocktail (Roche, Mannheim, Germany). Total protein was determined by the Bradford protein assay (Bio-Rad, CA, USA). Proteins were separated by 10% SDS/PAGE, transferred onto a PVDF membrane (Amersham Biosciences, UK) and then blocked with 10% non-fat milk. The membrane was then incubated with antibodies against 5-LOX (1:1000, BD Bioscience), GFAP (1:5000, Chemicon, Merck, USA, Cat. No. MAB3402), ED-1 (1:1000, Chemicon, Merck, Germany, Cat. No. MAB1435), OX-42 (1:1000, Chemicon, Merck, Germany, Cat. No. CBL1512), or β-actin (Cell Signaling Technologies, USA, Cat. No. mAb#4970) at 4 °C overnight, then washed and incubated in HRP-conjugated anti-rabbit or mouse IgG at room temperature for 1 h. Visualization was carried out using Luminata Forte or Crescendo Western HRP substrate (Millipore Corporation, USA) and chemiluminescence signals were detected using UVIchemi (UVItec, UK).