Rabbit anti human β actin

Cells were collected at different times. Cells were seeded in 10 cm dishes and then transfected and induced as described above. The cells were then lysed in a lysis buffer containing RIPA and an inhibitor cocktail (1:10). Cell lysates containing 30 μg of protein were resolved by SDS-PAGE and then transferred onto a polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA). The membrane was then blocked with 5% skim milk, followed by incubation with the following primary antibodies overnight at 4°C: rabbit anti-human Cav-1 (1:2000; Abcam), rabbit anti-human Lmx1a (2 μg/mL; Abcam), rabbit anti-human Nurr1 (1:100; Santa Cruz Biotechnology), rabbit anti-human TH (1:1000; Abcam) or rabbit anti-human β-actin (1:2000; Abcam). After four washes with Tris-buffered saline/Tween, the membrane was incubated with secondary antibody for 1.5 hours at room temperature. Protein signals were detected with the ECL kit (Beyotime, Shanghai, China) and quantitatively analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Total proteins were extracted from HDMECs and transfected HMEC-1 with RIPA lysis buffer (Solarbio, China), containing protease inhibitors, and protein concentrations were determined by the BCA kit (Solarbio, China). Protein lysates were carried out with ProteinSimple Wes (ProteinSimple, USA), an automatic protein expression quantitative analysis system for ultramicro samples, according to the manufacturer’s protocol. The following primary antibodies were used: rabbit anti-human ITGA9 (1:100, Abcam, UK) and rabbit anti-human β-actin (1:100, Abcam, UK). HRP-conjugated secondary antibody was goat anti-rabbit IgG (ProteinSimple, USA). β-actin was used as an internal standard, and expression levels of protein were normalized to β-actin.

Cells were lysed with radioimmunoprecipitation assay lysis buffer (1% NP-40, 0.5% Sodium deoxycholate and 0.1% SDS; Wuhan Boster Biological Technology, Ltd.) and protein concentration of whole cell lysates was determined using the Bicinchoninic Acid Protein Assay kit (Beyotime Institute of Biotechnology, Haimen, China). Briefly, 30 µg whole cell lysates were loaded and separated by electrophoresis on 10% SDS-polyacrylamide gels and were then transferred to polyvinylidene fluoride membranes (Beyotime Institute of Biotechnology). After blocking with 5% non-fat milk for 1 h at room temperature, the membranes were incubated with rabbit anti-human TIMP3 (cat no. ab39184; 1:1,000; Abcam, Cambridge, MA, USA) and rabbit anti-human β-actin (cat no. ab8227; 1:2,000; Abcam) overnight at 4°C. The membranes were then washed three times with TBST containing 0.1% Tween-20 and incubated with goat anti-rabbit secondary antibody (cat no. A0208; 1:1,000; Beyotime Institute of Biotechnology) for 1 h at room temperature. Subsequently, membranes were washed a further three times with TBST and were incubated with BeyoECL Plus chemical luminescence solution (Beyotime Institute of Biotechnology). The protein bands were visualized using a ChemiDoc XRS imaging system and were analyzed using Quantity One software version 4.3.0 (Bio-Rad Laboratories, Inc.).