Ecl system

Tissues containing ischemic penumbra were harvested for Western blotting at 24, 48, 72 h after MCAO following a standard protocol (Molecular Clone, Edition II). Briefly, the tissues were lysed in 300 μL lysis buffer (10 mM Tris, 150 mM NaCl, 1 % Triton X-100, 0.5 % NP-40, 1 mM EDTA, pH 7.4) and mixed with protease and phosphatase inhibitor cocktails (Roche). Nuclear protein was obtained using nuclear and cytoplasmic extraction reagents (Thermo Scientific). Twenty-five micrograms of cell lysate, as quantified with a BCA protein assay (Pierce), was separated on SDS-PAGE and transferred to PVDF membranes (Roche). The primary antibodies used were as follows: anti-glial fibrillary acidic protein (GFAP) (1:4000, Cell Signaling Technology), anti-Iba1 (1:1000, Abcam), anti-DRD2 (1:2000, Abcam), anti-p-STAT3 (Tyr705) (1:1000, Cell Signaling Technology), anti-STAT3 (1:1000, Cell Signaling Technology), anti-CRYAB (1:2000, Abcam), anti-β-actin (1:2000, Abcam), and anti-histone 3 (1:2000, Cell Signaling Technology). The secondary HRP-labeled antibodies were all from Cell Signaling Technology. Chemical reactions were detected with an ECL system (Advansta), and the scanned images were analyzed with ImageJ software (version 1.47). The protocols for cell culture experiments were the same as those described above.

To quantify the effect of PlGF on actin polymerization in EnSCs, cells were incubated with actin extraction buffer and processed for G and F-actin protein lysates^{22 (link)}. Bradford assay was performed to obtain protein concentration and equal volumes of each fraction were boiled with 1X Lamelli Buffer at 95 °C for 15 min. Proteins were separated on 10% SDS-polyacrylamide gels and transferred to PVDF membranes (#GE10600029, Amersham Biosciences). Non-specific binding sites were blocked by 1 h incubation at room temperature with 5% non-fat dry milk in TBS-T. The membranes were incubated overnight at 4 °C with primary antibodies against Pan-actin rabbit monoclonal antibodies (1:1000, #D18C11, Cell Signaling Technology). Post incubation with primary antibody, membranes were washed with TBS-T and incubated with HRP-conjugated anti-rabbit secondary (1:2000, #7074 s, Cell Signaling Technology) antibodies for 1 hour in room temperature. Antibody binding was detected post-developing with the ECL system (#R-03031-D25, Advansta) according to the manufacturer’s protocols. The fluorescence signals were obtained with an iBright CL1000 (ThermoFisher Scientific), and the intensities were assessed by a densitometry analysis to measure the relative expression of the pan- actin by ImageJ software.

Proteins were separated by SDS‐PAGE electrophoresis, which was performed on 10% or 12% of polyacrylamide gels, and then electro‐transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% of non‐fat milk and then incubated with different primary antibodies, including HIF‐1α, VEGF, p‐Erk, total Erk, p‐p38, total P38, p‐JNK, total JNK, β‐actin, followed by horseradish–peroxidase‐labelled secondary antibody. Immunoreactive proteins were detected using chemiluminescence ECL system (Advansta, Menlo Park, CA, USA), as described previously 19.

Total protein was extracted from the cells cultured under nitrogen starvation conditions for 0 and 24 h as described above. Protein concentration was measured using the BCA protein assay kit (Beyotime Biotechnology). The constant protein amounts (30 μg) from different conditions were separated by a 12% SDS-PAGE gel and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore) for western blotting. The membrane was blocked in 5% bovine serum albumin BSA for 2 h at room temperature and then incubated with primary antibodies (anti-mono/di-methyllysine, anti-trimethyllysine, anti-acetyllysine, anti-propionyllysine, anti-phosphotyrosine, anti-glutaryllysine, anti-2-hydroxyisobutyryllysine, and anti-crotonyllysine) at 1:2,000 dilution overnight at 4°C. After washing three times with Tris-buffered saline with Tween 20 (TBST) buffer (25 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.1% Tween 20), the membrane was continually incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1:5,000 dilutions) at room temperature for 1 h. The antibody response signal was monitored by ECL system (Advansta Inc., Menlo Park, CA).

Total protein was extracted from the cells cultured under nitrogen starvation conditions for 0 and 24 h as described above. Protein concentration was measured using the BCA protein assay kit (Beyotime Biotechnology). The constant protein amounts (30 μg) from different conditions were separated by a 12% SDS-PAGE gel and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore) for western blotting. The membrane was blocked in 5% bovine serum albumin BSA for 2 h at room temperature and then incubated with primary antibodies (anti-mono/di-methyllysine, anti-trimethyllysine, anti-acetyllysine, anti-propionyllysine, anti-phosphotyrosine, anti-glutaryllysine, anti-2-hydroxyisobutyryllysine, and anti-crotonyllysine) at 1:2,000 dilution overnight at 4°C. After washing three times with Tris-buffered saline with Tween 20 (TBST) buffer (25 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.1% Tween 20), the membrane was continually incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1:5,000 dilutions) at room temperature for 1 h. The antibody response signal was monitored by ECL system (Advansta Inc., Menlo Park, CA).