Protease and phosphatase inhibitor mixture

As described previously,22 cells were lysed with RIPA buffer (Beyotime, Shanghai, China) that contained a protease and phosphatase inhibitor mixture (Roche, Mannheim, Germany) and cells membrane protein were extracted by membrane and cytosol protein extraction kit(P0033; Beyotime). Protein extraction (50 μg) were boiled and subjected to a 10% SDS‐PAGE gel followed by immunoblotting with specific antibodies.

Properly anesthetized mice were decapitated to obtain the right-side brain tissues at 7 days after MCAO, after which, tissues were rapidly extracted on ice, weighed, and ground in liquid nitrogen. Then, ~20 mg of homogenate was weighed. The samples were resuspended in 50 μL of PBS and then incubated in 200 μL of a solution containing 1.5 mg·mL⁻¹ of a sulfo-NHSSS-biotin moiety (Pierce) at 4°C for 30 min to biotinylate surface proteins. Unreacted biotin was then quenched and removed with 100 mM glycine in PBS. After centrifugation (1,000 × g, 10 min, 4°C), the cell membranes were lysed in homogenization buffer containing 1% SDS and a protease and phosphatase inhibitor mixture (Roche). Biotinylated proteins were precipitated with 200 μL of Pierce avidin agarose for 2 h at 4°C. The agarose beads were precipitated by sequential centrifugation (500 × g, 3 min) followed by three washes with homogenization buffer. After the final precipitation, the washed beads were eluted twice with sample buffer and heated for 10 min at 98°C.

Proteins were purified in RIPA lysis buffer containing a protease and phosphatase inhibitor mixture (Roche Diagnostics). Protein concentrations were tested via a BCA assay kit (Pierce Diagnostics). Proteins were separated on SDS/PAGE gels, transferred to polyvinylidene difluoride membranes (Millipore), blocked in 5% skim milk (OXOID) in TBST (0.02 M Tris base, 0.1% Tween 20, 0.14 M NaCl pH 7.4), and incubated with primary antibodies overnight at 4°C. The primary antibodies used were anti-uncoupling protein 1 (UCP1) (ab209483, Abcam) and anti-HSP90 (4874, Cell Signaling Technology). Goat anti-Rabbit IgG H&L (A0277, Beyotime). The primary antibody was diluted at a ratio 1:1000; The secondary antibody was diluted at a ratio 1:5000. Signals were detected with super signal west pico chemiluminescent substrate (Pierce). Intensity values of the bands were analyzed via ImageJ software (National Institutes of Health, Bethesda, MD, USA).

The cytoplasmic and nuclear fractions separated using the protein extraction Kit (BioVision, Palo Alto, USA) according to the manufacturer’s instructions. Whole cell lysates were prepared in a buffer containing protease and phosphatase inhibitor mixture (Roche Molecular Biochemicals, Mannheim, Germany) at 4 °C. Western blotting analysis was performed as previously described^{20 (link)}. Primary antibodies were used: HMGB1, RAGE, β-actin, GAPDH, Lamin B1, phospho-ERK, ERK, and γH2AX (Cell Signaling Technology, USA, except HMGB1 and RAGE from Abcam, USA).

Whole-cell lysates were prepared using RIPA buffer containing a protease and phosphatase inhibitor mixture (Roche Applied Science). The extracted proteins were separated by SDS-PAGE and then transferred onto a nitrocellulose membrane (Bio-Rad). After blocking for 1 hr at room temperature with 5% non-fat milk (Bio-Rad), membranes were incubated overnight at 4°C with the indicated antibodies. Following incubation with an appropriate secondary antibody for 1 hr at room temperature, bound antibodies were detected with an ECL WB Detection System (GE Healthcare). Apoptosis was evaluated by measuring cleaved Caspase-3 (Cell Signaling Technology), PARP-1 and β-Catenin (Abcam) levels. β-actin (Santa Cruz Biotecnology) was used as a loading control.