Ab97069

Western blot was performed with the standard protocols. Protein was prepared from zebrafish embryos at 44 hpf and dissolved in 4 × protein SDS–polyacrylamide gel electrophoresis Loading Buffer (Takara). Nkx2.5, Stat1a and Hdac3 were detected with anti-Nkx2.5 (PA5-49431, Thermo Fisher, US), anti-Stat1a (SAB3500364, Sigma, US) and anti-Hdac3 (ab32369, Abcam, US) at the dilution of 1:1,000, followed by incubation with an anti-rabbit IgG-horseradish peroxidase antibody (ab97069, Abcam, US) at a dilution of 1:5,000. β-actin was used as the internal control using an anti-β-actin antibody (A2228, Sigma, US), followed by an anti-mouse IgG antibody (62-6520, Invitrogen, US) diluted 1:1,000 and 1:5,000 in the block solution, respectively. Full scans of all western blots are presented in Supplementary Fig. 10.

TWOD consists of red ginseng, Astragalus, Panax, leech, earthworm, Salvia, and Poria (weight ratio 2 : 15 : 2 : 3 : 2 : 10 : 15). All ingredients of the TWOD were combined and boiled for 2 hours. After boiling, TWOD was filtered and concentrated to 1 g/ml. Then through sterilization and vacuum packaging, TWOD was stored at 4°C at the China-Japan Friendship Hospital Extracting Room.
CaD was purchased from Xi'an Lijun Pharmaceutical Co. Ltd. Anti-occludin (ab31721), anti-claudin-5 (ab53765), anti-MMP-9 (ab137867), and horseradish peroxidases (HRP, ab97069) were purchased from Abcam. The antibody diluent (s3022) was obtained from DAKO. Trypsin (No. 0458) was get from Amresco. STZ (s0130) was purchased from Sigma.
The TWOD group was treated orally with TWOD, which was diluted in 5 ml of distilled water at a daily dose of 10 g/kg and considered as a therapeutic agent for the prevention of DR. The CaD group was used as the positive control group and was treated orally with CaD at the daily dose of 250 mg/kg diluted with 5 ml of distilled water. The control group and model group were treated with 5 ml distilled water over the same treatment period. The rats receive TWOD and CaD for 9 months.

Protein was isolated from cells as previously described (38 (link)) and was quantified using the Pierce Microplate BCA protein assay kit - reducing agent compatible (Thermo-Scientific), run on Bio-Rad Any-kDa Mini-Protean TGX precast gels, and transferred to nitrocellulose membranes in the Bio-Rad Midi Transfer Packs using the Bio-Rad Trans-Blot turbo blotting system. The membranes were blocked 1 h in 5% milk, before being probed overnight at 4 °C with SOX9 antibody (1:800, Abcam catalog no. ab26414). Secondary antibody was applied for 2 h following wash steps at the following dilutions: goat α-rabbit (1:4000, Abcam ab97069). Preconjugated β-actin-HRP (1:40,000, Sigma-Aldrich catalog no. A3854) was applied for 20 min and used as loading control. Western blot densitometry analysis was done using ImageJ.