Protein extraction kit

Total protein and cytosolic and nuclear protein in the ischemic cortex tissue were extracted with the corresponding protein extraction kit (Key GEN Biotech, China) following the manufacturer's protocols, and the protein quantity was assessed with the BCA protein assay kit (Key GEN Biotech, China). Equal protein quantities were separated using sulfate polyacrylamide gel electrophoresis (8%–12%), prior to transfer to polyvinylidene difluoride membranes (Millipore, USA). After that, they were blocked for 2 h in 5% milk before being exposed to HMGB1, TLR4, Bax, Bcl‐2, NF‐κB p65, Cleaved Caspase‐3, GAPDH and Lamin B antibodies for an entire night at 4°C. The blots were thrice rinsed for 10 min each, before 1 h incubation in matched horseradish peroxidase‐conjugated secondary antibody at room temperature 2 h. Following three more washes in TBST buffer, protein visualization was done on X‐ray film employing the Super ECL Plus Detection Reagent (Key GEN Biotech, China). Protein band quantification was done via Image J.
Significantly, during the operation, the ipsilateral ischemic cerebral cortex tissues were obtained, respectively, both were 1 cm³ in size. Each sample was divided into two parts. One part was stored in liquid nitrogen and prepared for protein and total RNA, whereas the other part was fixed in 4% buffered formalin and prepared for the histomorphological analysis.

A protein extraction kit (KeyGEN, Nanjing, China) was used to lyse the cells. Protein concentrations were measured with a BCA kit (Beyotime, Shanghai, China). The proteins were separated by SDS–PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk for 1 h at room temperature and was then incubated with antibodies against AMPK, p-AMPK (Thr172), mTOR, p-mTOR (Ser2448), NF-κBp65, phospho-NF-κBp65 (Ser536), p38 MAPK, phospho-p38 MAPK (Thr180/Tyr182), p70S6 kinase, phospho-p70 S6 kinase (Thr389), AKT, phospho-AKT (Ser473), Smad2, phospho-Smad2 (Ser465/467), β-actin, Beclin-1, LC3B, SQSTM1/p62 (Cell Signaling Technology, Beverly, MA, USA), fibronectin, α-smooth muscle actin (α-SMA), GAPDH, and human collagen (type I) (Thermo Fisher Scientific, Rockford, IL, USA) at 4 °C overnight. The membrane was then incubated with a goat anti-rabbit secondary antibody or a horse anti-mouse secondary antibody (Cell Signaling Technology, Boston, MA, USA) at room temperature for 1 h. Finally, the signals were examined using a chemiluminescence kit (Bio–Rad, Hercules, CA, USA).

Total protein was extracted from cells and uterine tissue with a protein extraction kit (KeyGEN, Changchun, China) according to protocols from the supplier. Protein concentration was determined using a bicinchoninic acid assay (KeyGEN). Equal concentrations of total protein were separated by SDS-PAGE (12%). Proteins were transferred onto polyvinylidene difluoride membranes and were blocked for two hours with TBST (50 mmol/L Tris, pH 7.6, 150 mmol/L NaCl, and 0.1% Tween 20) containing 5% BSA. The membranes were incubated with primary antibodies diluted in TBST overnight at 4 °C. Antibodies against cytochrome C (ab133504), caspase-3 (ab184787), BAX, (ab32503), and Bcl-2 (ab182858) were from Abcam (Shanghai, China). After washing three times in TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for two hours at room temperature and then washed three times for 10 min. Protein bands were visualized by exposure to an enhanced chemiluminescence detection system imager (Tanon Biotech, Shanghai, China) with an enhanced chemiluminescence solution (DiNING, Beijing, China). The relative intensity of each band was assessed by Image J 1.47 v software.

A protein extraction kit (KeyGEN, Nanjing, China, #KGP250, #KGP950) was used to lyse the cells. Protein concentrations were measured with a BCA kit (Beyotime, Shanghai, China, #P0010). Protein separation and detection were performed using an automated capillary electrophoresis system (Simple Western system and Compass software; ProteinSimple, San Jose, CA, USA, Version: 5.0.0). Wes Separation Capillary Cartridges for 12–230/66–440 kDa (ProteinSimple, San Jose, CA, USA, #SM-W004/SM-W008) were used for the proteins. The following primary antibodies were used: GAPDH (#5174S), α-SMA (#19245S), FN1 (#26836S), COL1A1 (#39952S), CTGF (#86641S), Smad2 (#5339S), phospho-Smad2 (p-Smad2, #3108S), Smad3 (#9523S), phospho-Smad3 (p-Smad3, #9520S), ERK (#4695S), phospho-ERK (p-ERK, #4370S), and Snail (#3879S) (all from Cell Signaling Technology, Boston, MA, USA). Signals were detected with an HRP-conjugated anti-rabbit secondary antibody (ProteinSimple, San Jose, CA, USA, #DM-001) and were visualized using Compass for SW software.