Rabbit anti human nurr1

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).

hADSCs were differentiated in 24-well chambers at 29 days, rinsed three times with PBS, and incubated in 4% paraformaldehyde overnight at 4°C. Bovine serum albumin (1%) in PBS was used for blocking. Afterwards, the cells were incubated overnight at 4°C with the following primary antibodies: mouse anti-human Cav-1 (1:100; Cell Signaling Technology), rabbit anti-human tyrosine hydroxylase (TH) (1:100; Abcam), rabbit anti-human Lmx1a (1:100; Abcam) or rabbit anti-human Nurr1 (1:100; Santa Cruz Biotechnology). The samples were then rinsed three times thoroughly with PBS and incubated with the following secondary antibodies: anti-rabbit IgG-FITC (Sigma-Aldrich), anti-mouse IgG-FITC (Abcam), anti-mouse IgG-TRITC (Sigma-Aldrich) or anti-rabbit IgG-TRITC (Abcam) for 1.5 hours at room temperature. The cells were thereafter rinsed three times in PBS and incubated for 5 minutes with Hoechst 33258. The samples were then washed twice with PBS and once in deionized water. Stained cells were observed under a confocal laser scanning microscope (SP8, Leica).