P mlc ser19

Cells were extracted from collagen gels by the action of Liberase TL (Roche) for 1 h at 37 °C. After having been lysed and dosed, protein samples were separated using SDS-PAGE and transferred onto nitrocellulose membranes in a transfer buffer (25 mM Tris, 200 mM glycine, Ethanol 20%). The blots were blocked with 5% low-fat milk in Tris-buffer saline (65 mM Tris pH 7.4, 150 mM NaCl) at room temperature for 1 h. They were incubated overnight with primary antibodies at 4 °C: p-p44/42 MAPK (Thr202/Tyr204) (E10, 1:1000), p-MLC Ser19 (#3671, 1:500), p-P70S6K Thr389 (1A5, 1:500, Cell signaling), β-actin (AC-15, 1:1000, Sigma-Aldrich), HSC-70 antibody (B-6, 1:5000, Santa Cruz Biotechnology). After being washed with TBS, the blots were incubated for 1 h with an HRP secondary antibody (1:1000) in 5% low-fat milk in TBS at room temperature. The blots were then washed with TBS. Immunocomplexes were visualized with a ChemDoc MP Imaging System (Bio-Rad) after a chemiluminescent reaction using the Ommobilon Western Chemiluminescent HRP substrate (Merck Millipore).

Cardiomyocytes were dissociated by TrypLE around day 10, and different doses of nicotinamide or ROCK inhibitor Y27632 10 μM were added in the medium for 1 h. The protein was harvested after 1 h for western blot [22 ]. Briefly, 40 μg protein of each sample was separated by electrophoresis with SDS-PAGE gels, and then transferred to PVDF membranes. The membranes were first blocked with 5% non-fat milk, and then incubated with antibodies against p-MLC (Ser19) 1:500 (Cell Signaling, 3671), MLC 1:1000 (Sigma, M4401), or Actin 1:2000 (Santa Cruz, sc-47778) overnight at 4 °C. After washing, the membranes were incubated with HRP-secondary antibodies (Jackson, 115-035-146 or 111-035-144) for 2 h at room temperature. Chemiluminescence was detected using SuperSignal^TM West Pico PLUS Chemiluminescent Substrate (Thermo) or West Dura Extended Duration Substrate (Thermo).

Cells seeded on top of a thick layer of collagen I were fixed with p-formaldehyde, permeabilized with 0.3% Triton-X 100 (v/v), and blocked with 4% BSA in PBS for 30 minutes. Cells were then incubated with primary antibody (pMLC Ser19, Cell Signaling) and stained with secondary Alexa Fluor-647 anti-rabbit (Life Technologies) and Alexa Fluor 546-phalloidin for F-actin detection (Life Technologies). Multisite bright-field microscopy of cells in 24-well plates containing thick layers of collagen was performed (8 (link)). Further details are given in the Supplementary Materials (available online).