Supersignal west pico maximum sensitivity substrate

Lysates from aortic VSMCs were prepared in 8 M urea and run under denatured conditions on an SDS-PAGE 4%–12% gradient gel, transferred onto nitrocellulose membranes, and blocked and incubated overnight at 4°C with primary antibodies. Immunocomplex detection was performed with the enhanced chemiluminescence SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) and SuperSignal West Pico Maximum Sensitivity Substrate (Thermo Fisher Scientific) using the ChemiDoc XRS+ Molecular Imager (Bio-Rad). Densitometry analysis was performed using ImageLab Software. Information on antibodies used is available in Supplemental Methods.

Myosin light chain2 (MLC2) is also known as myosin regular light chain (MRLC). ROCK could phosphorylate ser19 of MLC2 of smooth muscle. ROCK inhibitors could lower IOP by increasing aqueous humor outflow through the trabecular meshwork (TM). The A7r5 cell (Rat muscle cell, ATCC CRL-1444) was purchased from American Type Culture Collection (Manassas, VA, USA) and was maintained in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin antibiotics at 37 °C in a humidified atmosphere of 5% CO₂. Cells were seeded at 6*10⁵ cells per well in a P100 dish and incubated overnight.
Cells were changed to a new medium prior to treatment. Cell lysis and Western blots were performed by following the usual protocol. CCME prepared in 100% DMSO as a stock solution was added to cell lysate at various concentrations. AR-13324 was used as the positive control. The cell lysate was assayed with pMLC2 (Thr19/Ser19) antibody (Cell Signaling, CST3674) in HaltTM protease and phosphatase inhibitor (Thermo Fisher 78442, Waltham, MA, USA). GAPDH (Sigma), HRP goat anti-rabbit (Jackson Immuno Research, 111-035-003, West Grove, PA, USA), skim milk, and SuperSignal West Pico Maximum Sensitivity substrate (Thermo Fisher, 34080) were used for the Western blot.