Ab138498

Mouse Trim59, Trim27, WASH, and control siRNAs were purchased from Ribo, Shenzheng, China. Anti- Trim59 (ab69639, Abcam), anti-Trim27 (A6405, Abclonal), anti-WASH (SAB-4200552, Sigma), anti-Oct4 (ab18976, Abcam), anti-FLAG (M20008, Abmart), anti-HA (#T501-1, Signalway Antibody), anti-Ub (YT4793, Immunoway), anti-K48 (#4289, Cell Signaling Technology), anti-K63 (BML-PW0600, Enzo), anti-ACTR2 (ab134082, Abcam), anti-MYH9 (ab138498, Abcam), anti-CAPZA1 (ab166892, Abcam), anti-CAPZB (ab175212, Abcam), anti-β-actin (SC-81178, Santa), Phalloidin-iFluor 488 (ab176753, Abcam), DAPI (#4083, Cell Signaling Technology), Alexa Fluor 488 Conjugate (#4416, #4408, Cell Signaling Technology), and Alexa Fluor 594 Conjugate (#8890, #8889, Cell Signaling Technology) antibodies were purchased.
Murine FL Trim59 clone was obtained from the ATCC. Trim59 mutants were constructed by performing PCR with four primers according to a previous method^{33 (link)}. All primers used in this study are listed in supplementary Table S2b. YFP–WASH and YFP-WASH K220R mutant were from Patrick Ryan Potts, UT Southwestern Dallas, TX 75390, USA; plasmids encoding hemagglutinin (HA)-tagged ubiquitin (HA-Ub), HA-K48-Ub, and HA-K63-Ub were obtained from Y. Xiong (University of North Carolina, Chapel Hill).

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.

Immunofluorescence analysis was performed as previously described.³³ Briefly, 4% paraformaldehyde was used to fix attached human NP cells, then 0.2% Triton X‐100 in PBS was used to permeabilize. The slides were washed in PBS and blocked with 2% bovine serum albumin (BSA) in PBS for 2 hours at 37°C and then incubated with primary antibodies against: myosin IIA (Abcam, ab138498, 1:200), myosin IIB (Abcam, ab230823, 1:100), MRTF‐A (Abcam, ab115319, 1:200). After washed twice, the slides were subsequently treated with secondary goat anti‐rabbit antibody (Boster) at 37°C for 2 hours. Nuclei were co‐stained for 5 minutes with 0.1 g/mL DAPI (Beyotime, Nantong, China), and images were captured under a microscope (Olympus, BX53). To analyse the nuclear‐to‐cytoplasmic fluorescent intensity ratio of MRTF, the nuclear intensity and cytoplasmic intensity of 20 cells from each group were calculated and analysed by Image J (NIH). For co‐localization analysis of myosin II isoforms with F‐actin, the Pearson coefficient was calculated by Image J (NIH) and the Coloc 2 plugin.