Immobilon polyvinylidene fluoride membrane

To detect the expression levels of CCL5 in stroke mice, Western blot was performed as previously described.23 Samples were harvested 72 hours after ischemia, gently homogenized in cold lysis buffer (10 mmol/L 4‐2‐hydroxyethyl‐1‐piperazine‐ethanesulfonic acid [pH 7.9], 1.5 mmol/L MgCl₂, 10 mmol/L KCl, 1 mmol/Ldithiothreitol) and protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN), and then centrifuged. The supernatants were collected and used for analysis. Protein concentrations were measured using bicinchoninic acid (Pierce Chemicals, Rockford, IL). Proteins (36 μg) were loaded into each lane, separated with 4% to 15% polyacrylamide gels (Bio‐Rad, Hercules, CA), and electrotransferred to Immobilon polyvinylidene fluoride membranes (Millipore Corp, Billerica, MA). Membranes were blocked with 5% nonfat dry milk in phosphate‐buffered saline (PBS) with 0.1% Tween 20 for 1 hour, and incubated with 1:250 anti–mouse CCL5 antibody (R&D Systems Inc, Minneapolis, MN) overnight at 4°C. They were then washed 3 times with 0.1% Tween in PBS, then incubated with 1:1000 secondary antibody (Cell Signaling, Danvers, MA) for 2 hours at room temperature, and washed 3 times with 0.1% Tween in PBS. Signals were detected with ECL reagent (Amersham, Piscataway, NJ). β‐Actin was used as an internal control. Densitometric analysis of bands was performed with Image J software.

Total protein was extracted with pro-prep for 30 min on ice and vortexed for 5 s every 5 min. The supernatant was collected and the protein concentration was determined using a bicinchoninic acid kit (Thermo Fisher Scientific, Rockford, IL, USA). Proteins were separated by 8%–10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to immobilon polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk at room temperature for 1 h, and incubated with primary antibodies against AKT, p-AKT (S473) (Cell signaling technology, Boston, MA, USA), β-actin (Santa Cruz Biotechnology, Dallas, TX, USA) at 4°C overnight. After removing the primary antibody, the membranes were washed with 1× Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h. The membranes were incubated with secondary antibodies at room temperature for 1 h. Anti-rabbit horseradish peroxidase (HRP) conjugate and anti-mouse HRP conjugate (Enzo Life Sciences, Farmingdale, NY, USA) were used as secondary antibodies. After washing the membranes again with 1× TBST for 1 h, the bands were visualized using immobilon crescendo western HRP substrate (Immobilon; Millipore) and X-ray film. β-actin was used as the loading control for Western blot results.

The placentae were homogenized using a Tissue-Tearor (RPI Corp., Mt. Prospect, IL) in a lysis buffer. Protein aliquots were separated in SDS-PAGE gels, transferred to Immobilon-polyvinylidene fluoride membranes (Millipore, Billerica, MA) and then incubated overnight with antibodies as listed in Supplemental Table S2. Peroxidase-conjugated secondary antibody (1:2000 dilution; Vector Laboratories, Burlingame, CA) was used as the secondary antibody. Immunoblotting signals were detected by the enhanced chemiluminescence (ECL) Western blot detection system (GE healthcare Biosciences, Pittsburgh, PA). All membranes were reblotted with β-actin antibody as the loading control. The intensity of specific bands was scanned using image analysis software TotalLab. The results were presented as the ratio of target protein over β-actin.