Ab9485

Proteins were extracted using RIPA Lysis Solution (P0013C; Beyotime), and the protein concentration was measured using the BCA protein kit (KeyGen Biotech Co., Ltd). The proteins were separated by using SDS‐PAGE and transferred onto polyvinylidene fluoride membranes. The membrane was then incubated with the following primary antibodies: MMP‐13 (1:1000; 18165‐1‐AP, Proteintech), IL‐6 (1:500; 21865‐1‐AP, Proteintech), TNF‐α (1:500; 17590‐1‐AP, Proteintech), JAK1 (1:1000; ab133666, Abcam), p‐JAK1 (1:1000; ab138005, Abcam), STAT3 (1:1000; ab68153, Abcam), p‐STAT3 (1:1000; #9131; Cell Signaling Technology), Wnt1 (1:1000; ab15251, Abcam), β‐catenin (1:500; ab223075, Abcam), and GAPDH (1:1000; ab9485, Abcam). The next day, the membranes were incubated with the secondary antibody, and the intensity of protein expression was detected using ECL chemiluminescence (Beyotime).

Proteins from samples were isolated using RIPA buffer (Vazyme, Nanjing, China) and were segregated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then proteins were transferred onto the polyvinylidene difluoride membranes (Vazyme). The membranes were blocked with 5% skimmed milk (Vazyme). Thereafter, the membranes were incubated with the primary antibodies: anti-Irak2 (1:2000, ab62419, Abcam, Cambridge, UK) or glyceraldehyde 3-phosphate dehydrogenase (1:2500, ab9485, Abcam) overnight. After being rewashed, the membranes were incubated with the secondary antibody (1:3000, ab205718, Abcam) for 2 h. The membranes were analyzed by the ChemiDoc™ MP Imaging System (Bio-Rad) after being treated with ECL kit (Vazyme).

RIPA buffer (Thermo Scientific) containing Complete EDTA-free protease inhibitor was used to isolate proteins from cells and tissues. Proteins (30 μg) were loaded with a 4–15% TGX gel (BioRad) and then transferred to 0.2 µm PVDF membranes (BioRad). The membranes were blocked in 5% non-fat dried milk/Tris-buffered saline and 0.1% Tween-20 for 1 h at room temperature. Next, the membranes were incubated with primary antibodies: anti-SIPL1 (Abcam, ab79039), anti-MAZ (Abcam, ab85725), anti-AKT (Abcam, ab18785), anti-p-AKT (Abcam, ab38449), anti-P65 (Abcam, ab32536) or anti-p-P65 (Abcam, ab76302), and anti-GAPDH (Abcam, ab9485) overnight, along with horseradish peroxidase-labeled IgG (Abcam, ab205718) at room temperature. The protein bands were detected using enhanced chemiluminescence (Pierce, Rockford, IL, USA). Densitometry analysis was performed using ImageJ software (version 1.36; National Institutes of Health, Bethesda, MD, USA).

Tissue samples from the hippocampus and prefrontal cortex of offspring were homogenized in extraction buffer (C500006, Sangon Biotech, Shanghai, China) according to the manufacturer’s instructions. Each sample was adjusted to a final protein concentration of 1 μg/μl, mixed with Laemmeli’s sample buffer, and boiled for 5 min. Samples (40 mg) were loaded onto 8% bisacrylamide gels and separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were electrophoretically transferred from gels to polyvinylidene fluoride (PVDF) membranes that were then incubated with the following primary antibodies: anti-BDNF (1:200 dilution, AV41970, Sigma-Aldrich, St. Louis, MO, USA), anti-SERT (1:200, AG1204, Abgent, San Diego, CA, USA), anti-CREB (1:500 dilution, ab31387, Abcam, Cambridge, MA, USA), anti-pCREB (1:500 dilution, ab32096, Abcam), and anti-GAPDH (1:2500 dilution, ab9485, Abcam). Dilutions of peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:2000 dilution, sc-2004, Santa Cruz Biotechnology, Dallas, TX, USA) were prepared following the manufacturer’s instructions. Immuno-positive bands were visualized using a chemiluminescent method (G:BOX chemiXR5, SYNGEN, Sacramento, CA, USA), and the band densities were determined with the Gel-Pro32 software [39 (link)]. All western blotting experiments were repeated at least three times.

Total protein was extracted followed by concentration measurement utilizing a bicinchoninic acid kit. Protein (50 μg) was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane which was then blocked with 5% skimmed milk powder at ambient temperature for 1 h. Next, the membrane was probed with diluted primary rabbit antibodies to Smad2 (ab33875, 1: 1000, Abcam), Smad4 (ab40759, 1: 5000, Abcam), HDAC3 (ab7030, 1: 500, Abcam), TGIF1 (ab52955, 1: 5000, Abcam), cleaved caspase-3 (ab32042, 1: 1000, Abcam), total caspase-3 (ab32150, 1: 1000, Abcam), MMP-2 (ab97779, 1: 2000, Abcam), MMP-9 (ab38898, 1: 1000, Abcam), TGF-βRII (sc-17791, 1: 1000, Santa Cruz Biotechnology, Santa Cruz, CA) and GAPDH (ab9485, 1: 2500, Abcam) overnight at 4ºC. The next day, the membrane was re-probed with HRP-labeled secondary antibody of goat anti-rabbit antibody to IgG H&L (ab97051, 1: 2000, Abcam) for 1 h. The results were visualized utilizing enhanced chemiluminescence reagents (BB-3501, Ameshame, Little Chalfont, UK). Images were acquired through Bio-Rad image analysis system (Bio-Rad Laboratories, Hercules, CA), followed by analysis using Quantity One v4.6.2 software. The relative protein level was described as the ratio of gray value of protein to be tested to that of GAPDH [22 , 23 (link)].