Rock1

The following antibodies were used for Western blot analysis: Drp1 (5391, 1:1,000 working dilution), cleaved-caspase 3 (9661, 1:1,000), cleaved-caspase 9 (9505, 1:1,000 ), Cox IV (4850, 1:2,000), phospho-cofilin (Ser 3) (3313, 1:500), phospho-Drp1 (Ser 637) (4867, 1:500), phospho-Drp1 (Ser 616) (3455, 1:500), LIMK1 (3842, 1:2,000), LIMK2 (3845, 1:1,000) and phospho-LIMK1(Thr508)/LIMK2(Thr505) (3841, 1:500) were purchased from Cell Signaling Technology; GAPDH (sc-25778, 1:500), cytochrome c (sc-13156, 1:1,000), cofilin (sc-376476, 1: 2,000) and PP1 (sc-7482, 1:1,000) were purchased from Santa Cruz Biotechnology; PARP (ab32071, 1:1,000) and ROCK1 (ab45171, 1:1,000) were from Abcam; PP2A (610555, 1:5,000) was purchased from BD Biosciences. Erucin (sc-204741) and Y-27632 (sc-216067) were purchased from Santa Cruz Biotechnology.

TRPV4, ROCK1, ROCK2, and paxillin (PXN) antibodies were purchased from Abcam (Cambridge, MA, USA). RhoA, RhoB, RhoC, CDC42, RAC1/2, LIMK, phospho-LIMK, cofilin, phospho-cofilin, MLC, phospho-MLC, MYPT, and phospho-MYPT antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). GAPDH were purchased from Proteintech (Rosemont, IL, USA). Rho pathway antagonist Y27632, calcium chelator BAPTA-AM, TRPV4 agonist GSK1016790A, and its antagonist HC067047 were purchased from Selleck (Shanghai, China). Opti-MEM medium and Lipofectamine RNAiMAX reagent, Fluo-4 AM, Rhodamine Phalloidin, puromycin, and DAPI were purchased from Invitrogen (Massachusetts, USA).

After adding 200 μL of the lysate to the 6-well plate, it was allowed to stand for 20 minutes on ice, and the liquid was collected and centrifuged (13,000 rpm, 15 min); then, the supernatant was collected. The concentration of the protein was determined by the bicinchoninic acid (BCA) (Camilo Biological, Nanjing, China) method and quantified. The protein was separated using a 10% sodium dodecyl sulfate-polyacrylamide gel, then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA) for 2 hours at 4°C. 5% skim milk was prepared with Tris-buffered saline with Tween-20 (TBST) to block the specific antigen for 2 hours. After washing with TBST for 1 minute, PVDF membranes were incubated with a specific primary antibody (SOD1, Abcam, Rabbit; 1 : 3000; SOD2, Abcam, Rabbit, 1 : 3000; GPX1, US 1 : 1000, CST; GPX3, US 1 : 1000, CST; Bcl-2, Abcam, Rabbit, 1 : 2000; Bax, Abcam, Rabbit, 1 : 500; Sirt1, Bioworld, mouse, 1 : 500, China; P53, Abcam, Rabbit, 1 : 2000; ROCK1, Abcam, Rabbit, 1 : 500; ROCK2, Abcam, Rabbit, 1 : 1000; MYPT-1, Abcam, Rabbit, 1 : 1000; GAPDH US 1 : 1000 CST) at 4°C overnight. The next day, TBST was washed for 30 minutes. Specific proteins were detected by secondary antibodies and observed by the electrochemiluminescence (ECL) (Pierce, Rockford, IL, USA) system.

Total protein was extracted from HUVECs and lysed in RIPA buffer containing phosphatase inhibitor cocktail (Bimake) and protease inhibitor cocktail (MCE). Proteins were added to the wells of an SDS-PAGE system, separated by 10% SDS–PAGE and transferred to a PVDF membrane. After blocking in 5% milk for 1 h at room temperature, the membrane was incubated with β-actin (Servicebio), FGFR1 (CST), ROCK2 (Abcam), ROCK1 (Abcam), ERM (CST), MYPT1 (CST), pROCK2 (Abcam), pROCK1 (Abcam), pERM (CST), pMYPT1 (Invitrogen), GAPDH (Servicebio), ICAM1 (Servicebio) and VCAM1 (Abcam) at 4°C overnight and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit or mouse IgG at room temperature for 1 h. Bands were visualized using the ChemiDoc Imaging System (TOUCH IMAGER™).

The B3 cells were cultured in 24-well plates and treated with 200 µM H₂O₂ in media for 24 h. These cells were fixed with 4% paraformaldehyde for 10 min, permeabilized in 0.3% Triton X-100 for 10 min, blocked with 3% bovine serum albumin (BSA) for 30 min at 37 ℃. The primary antibodies of ROCK1 (1:200, Abcam) and p53 (1:200, Cell Signaling Technology) were first incubated at 37 ℃ for 1 h and then at 4 ℃ overnight. Alex Fluor 594 goat anti-rabbit.
IgG (Invitrogen, A11037) and Alex Fluor 488 goat anti-mouse IgG (Invitrogen, A11029) were as the secondary antibody incubated for 1 h at 37 ℃. Images were selected using a fluorescence microscope.

Total protein extracts (30 μg) was separated on 8–12% Bis-Tris gels, followed by transferring onto polyvinylidene fluoride membranes. After blocking with 5% skimmed milk for 2 h at room temperature (RT), the blots were incubated with a primary antibody overnight at 4 °C and then with horseradish peroxidase-conjugated secondary antibody for 1 h at RT. The primary antibodies included ROCK1 (Abcam, Cambridge, UK; 1:1000), RhoA (Abcam; 1:1000), and dynmin-related protein-1(Drp1) (Abcam; 1:1000). The intensities of protein bands were visualized using chemiluminescence with an ImageQuant 350 (GE Healthcare, Woburn, MA, USA). GAPDH was used as a loading control.