Nuclear cytosol extraction kit

Total protein was extracted from human NP cells by RIPA lysis buffer containing protease inhibitor (Beyotime). Nuclear and cytoplasmic proteins were extracted from NP cells by a Nuclear‐Cytosol Extraction Kit (Solarbio) according to the manufacturer's instructions. Western blot procedure was performed as previously described.³³ Primary antibodies against the following proteins were used: myosin IIA (Abcam, ab138498, 1:1000), myosin IIB (Abcam, ab230823, 1:1000), p21 (CST, 2947, 1:1000), p53 (Proteintech, 10442‐1‐AP, 1:1000), Cyclin D1 (Abcam, ab134175, 1:10 000), CDK4 (Abcam, ab108357, 1:1000), MRTF‐A (Abcam, ab115319, 1:1000), RhoA (Abcam, ab187027,1:1000), p‐RhoA‐Ser188 (Abcam, ab41435, 1:1000), ROCK1 (Proteintech, 21850‐1‐AP, 1:1000), ROCK2 (Proteintech, 20248‐1‐AP, 1:1000), MLC (CST, 3671, 1:1000), p‐MLC‐Ser19 (CST, 3671, 1:1000), GAPDH (Affinity, AF7021, 1:2000), Lamin B (Proteintech, 12987‐1‐AP, 1:1000). GAPDH and Lamin B were used for normalization. The experiments were repeated three times.

Nuclear and cytosol fractions were extracted from the mice midbrain tissues with Nuclear-Cytosol Extraction Kit (Solarbio, Beijing, China) following the manufacture’s protocols. Total proteins were isolated by RIPA lysis solution (Solarbio, Beijing, China). Protein levels were quantified using BCA assay. Equal amounts of protein (10–30 μg/lane) were separated on 10% Bis-Tris Nu-PAGE gel. Then, the membranes were incubated with primary antibodies: TH (1:2000), Iba-1 (1:800), GFAP (1:1000), Nrf2 (1:1500), Keap1 (1:2000), HO-1 (1:10000), NQO1 (1:2000), PCNA (1:2000), TNF-α (1:800), iNOS (1:1500), GDNF (1:800), BDNF (1:1000), and β-actin (1:4000). Next day, the membranes were incubated with anti-rabbit IgG secondary antibodies at 1:5000 for 1 h. Finally, the blots were detected using ECL substrate. β-actin was used as an internal standard to monitor loading errors. Densitometric analysis of immunoblots was performed using the Quantity One (Bio-Rad, Hercules, CA, USA) software system. The ratio of densitometry values of purpose protein with β-actin was analyzed and normalized to each respective control groups.