Ab41435

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.

NP samples were fixed in 10% formaldehyde for 24 hours and embedded in paraffin. Then, the samples were sliced into 4‐μm sections. Immunohistochemistry was carried out as described previously.³⁴ The sections were incubated with antibodies against: p‐RhoA‐Ser188 (Abcam, ab41435, 1:100), ROCK1 (Proteintech, 21850‐1‐AP, 1:100), ROCK2 (Proteintech, 20248‐1‐AP, 1:50), p‐MLC‐Ser19 (CST, 3671, 1:100). Staining was performed using the Dako REAL EnVision Detection System, Peroxidase/DAB+, Rabbit/Mouse (Dako Cytomation), according to the manufacturer's instructions. The sections were imaged by microscopy (Olympus).