Enhanced chemiluminescence ecl kit

The A549 cells were collected 60 h post of infection. The cells were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer with protease inhibitors (Beyotime, Shanghai, China) added to collect total proteins. The proteins were loaded onto SDS-PAGE gels (10–15%), electrophoresed, and then transferred to polyvinylidene difluoride (PVDF) membranes (CWBiotech, Beijing, China) through an electrophoretic transfer chamber (Millipore, Temecula, CA, USA). The membranes were washed with Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBST) three times and blocked with 5% nonfat milk in TBST at 37 °C for 2 h. Subsequently, the membranes were incubated with primary antibodies at 4 °C overnight and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature (Goat anti-Rabbit IgG: Abcam, ab205718, 1:5000 dilution). The primary antibodies used were: GAPDH: CST, 5174, 1:1000 dilution; IRF1: CST, 8478, 1:1000 dilution. Finally, the immunoblot bands were visualized with an enhanced chemiluminescence (ECL) kit (Beyotime, China) and read using a chemiluminescence system (Thermo Scientific, Waltham, MA, USA).

Streptavidin‐agarose bead–bound proteins were released in 5× loading buffer (Beyotime Biotechnology) at 55°C for 30 minutes. Total proteins and cytoplasmic proteins were boiled at 100°C for 10 minutes. Proteins were separated using a 10% SDS‐PAGE gel for 120 minutes (the amount of protein loaded per channel was 50 μg) and were then transferred to polyvinylidene‐difluoride membranes (0.45 μm pore size). An Anti‐EAAT2 antibody (Abcam) was used at 1:1000 (v/v), and a secondary antibody (Beyotime Biotechnology) was used at 1:1000 (v/v). An enhanced chemiluminescence (ECL) kit (Beyotime Biotechnology) was used for protein‐band exposure. The grey value of each protein band was quantified using ImageJ software to determine the total, membranous and cytoplasmic expression levels of EAAT2 and its mutants. The expression levels of total proteins, membranous proteins and cytoplasmic proteins were normalized to corresponding levels of actin (Beyotime Biotechnology), integrin β1 (Cell Signaling Technology), and α‐tubulin (Beyotime Biotechnology), respectively. Data were standardized and expressed as a percentage of protein expression compared with that of WT EAAT2.

Based on previous studies [48 (link)], the cultured primary cortical neurons and brain tissue surrounding the infarct were collected and lysed in Western lysis buffer (Beyotime Biotechnology Inc., Shanghai, China) on ice for 20 min. After centrifugation at 12000 r/min for 10 min, the supernatant was used to detect the protein concentration with an enhanced BCA Protein Assay Kit (Beyotime Biotechnology Inc.). Then, the protein samples (30 μg/lane) were loaded onto 10% SDS polyacrylamide gel for electrophoretic separation, and then transferred to a polyvinylidene difluoride (PVDF) membrane (IPVH00010; Millipore, Billerica, MA, USA). The membrane was immediately blocked with 5% bovine serum albumin (Biosharp, Hefei, China) for 1 h at room temperature. Then, the membrane was incubated overnight with the primary antibody at 4 °C, and with the HRP-conjugated secondary antibodies for 1 h at room temperature. Finally, the membrane was revealed with an enhanced chemiluminescence (ECL) kit (Beyotime Biotechnology Inc.). Additionally, β-tubulin and GAPDH were used as loading controls. Full length uncropped original western blots were shown in Supplementary materials 2.

The total proteins from C2C12 cells were isolated using RIPA buffer (Beyotime, Shanghai, China) and the protein concentration was measured using the BCA protein assay kit (Beyotime, Shanghai, China). The target proteins were separated using SDS-PAGE electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. Then, the PVDF membranes were incubated with specific primary antibodies overnight at 4 °C. Excess primary antibody on the PVDF membranes was washed away. Subsequently, the membranes were incubated with secondary antibody for 2 h at 37 °C. Finally, the protein bands were visualized using the enhanced chemiluminescence (ECL) kit (Beyotime, Shanghai, China) and detected using the Tanon-4600 (Tanon, Shanghai, China). Protein band intensities were quantified by Image J software and normalized against β-actin. The following primary antibodies were used: anti-MyoG (orb6492, Birobyt, Cambridge, UK; 1:200), anti-LC3 (83506, Cell Signaling Technology, Boston, MA, USA; 1:1000), anti-MAP4 (ab245578, Abcam, London, UK; 1:2000), and anti-β-actin (AF7018, Affinity, Changzhou, China; 1:5000). The immunoprecipitation analysis was performed as previously described [39 (link)]. Briefly, C2C12 cells were isolated with RIPA buffer and total proteins were immunoprecipitated with protein G beads. Western blotting was used to analyze the immunoprecipitated products.

Saw palmetto extract was purchased from Yongyuan Bio-technology, Co., Ltd. (Xi’an, China). Rabbit anti-B-cell lymphoma-extra large (Bcl-xL), anti-p53, anti-PI3K and anti-β-actin antibodies were purchased from Bioss, Inc. (Wuhan, China). An enhanced chemiluminescence (ECL) kit was obtained from the Beyotime Institute of Biotechnology (Shanghai, China).