Ecl developer

Proteins from transfected or treated cells were extracted and separated by SDS–PAGE before transferring to a nitrocellulose membrane, which was subsequently exposed to the appropriate primary antibody before detection using the horseradish peroxidase conjugated secondary antibody. Enhanced chemiluminescence (ECL) images were developed using ECL developer (Millipore, Darmstadt, Germany). Primary and secondary antibodies used are listed in the Additional file 1: Table S2.

The herbs were provided by Shanghai Huayu Chinese Herbs Co. Ltd. (Shanghai, China). Dihydroethidium (DHE) was purchased from Molecular Probes Inc. (Eugene, USA). Antibodies of p47phox, p67phox, p40phox, p22phox, and gp91phox were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Ras-1 and β-actin were obtained from Cell Signaling Technology (Danvers, MA, USA); eNOS and p-eNOS were provided by Abcam (Cambridge, MA, USA). Goat anti-rabbit secondary antibodies were bought from Wuhan Boster Biotech Co. Ltd. (Wuhan, China). ECL developer was from Millipore (Billerica, MA, USA). BCA protein quantity kits and PVDF membranes were provided by Pierce (Rockford, IL, USA). Phenylephrine (PE), acetylcholine (ACh), Hcy, sodium nitroprusside (SNP), tempol, apocynin, and N-nitro-L-arginine methyl ester (L-NAME) were purchased from Sigma Chemical Co. Ltd. (St. Louis, MO, USA). All reagents available were of high purity.

Bone marrow‐derived mesenchymal stem cells (BMSCs)s were collected and washed 3 times with PBS, with the addition of 100‐200 μL RIPA lysates (Beyotime Biotechnology, Shanghai, China), and cells were lysed in ice water by ultrasound. And the Bradford method was utilized to determine protein concentration. Equal amounts of protein from each group were subjected to 10% SDS‐PAGE electrophoresis, and the protein on the gel was transferred to PVDF membranes (Millipore, Bedford, MA, USA). For one hour, the membranes were sealed at 4°C, then they were incubated with the addition of Anti‐Smad7 antibody(ab216428), Anti‐p‐Smad2 antibody (ab18834), Anti‐Smad2 antibody (ab40855), Anti‐p‐Smad3 antibody (ab52903), Anti‐Smad3 antibody (ab40854), Anti‐Bcl2 antibody (ab182858) and Anti‐Bax antibody (ab32503) (overnight, 4°C). Next, TBST was used to wash the membranes twice. They were incubated with fluorescein‐labelled goat anti‐rabbit (ab205718, 1:2500) at room temperature for 1 hour. Abcam (Cambridge, UK) supplied the above antibodies. After being washed three times, they were exposed with ECL developer (Millipore, Bedford, MA, USA) and imaged with a membrane scanner.

The brain tissues, SH-SY5Y neurons and BV2 cells were harvested and then washed with cold PBS 3 times. Then, 100~200 μL RIPA lysate (Beyotime Biotcchnology, Shanghai, China) was added to lyse the cells in ice water, and Bradford method was adopted to determine the protein concentration. Afterward, an equal volume of protein from each group was loaded on 10% SDS-PAGE electrophoresis, and the protein on the gel was transferred to PVDF membranes (Millipore, Bedford, MA, USA). Subsequently, the membranes were blocked at 4° C for 1 hour, and the primary antibody (concentration 1: 1000) Anti-FSTL1 antibody (ab11805), Anti-β-actin antibody (ab115777), Anti-p-NF-κB antibody (ab86299), Anti-NF-κB antibody (ab16502), Anti-NLRP3 antibody (ab214185), Anti-ASC antibody (ab180799), Anti-Caspase-1 antibody (ab74279), Anti-IL-1β antibody (ab7632), Anti-Caspase3 antibody (ab13847), Anti-Bcl2 antibody (ab182858) and Anti-Bax antibody (ab32503) were added for incubation overnight at 4° C. After that, the membranes were washed twice with TBST at room temperature, and incubated with fluorescein-labeled secondary antibody goat anti-rabbit (ab205718, 1: 2500) for 1 hour at room temperature. The above antibodies were obtained from Abcam, MA, USA. After being washed 3 times, the membranes were exposed with ECL developer (Millipore, Bedford, MA, USA) and imaged with a membrane scanner.