Luminol enhancer solution

Cells were lysed in cell lytic M buffer (Sigma, St. Louis, USA) supplemented with 0.1% phosphatase-inhibitor (Sigma, St. Louis, MO, USA) and 0.1% protease-inhibitor (Sigma, St. Louis, MO, USA). Isolated proteins (40 μg) were fractioned using 12% SDS gels and electro-transferred to a polyvinylidene difluoride membrane (Merck Millipore, Cork, Ireland). Primary antibodies against S100A4 1:250 (HPA007973; Sigma, St. Louis, USA), CYR61 1:250 (HPA029853; Sigma, St. Louis, MO, USA), YAP 1:250 (sc-398182; Santa Cruz Biotechnology, Dallas, TX, USA), ERK1/2 1:1000 (4695S;Cell Signaling Technologies Inc., Danvers, MA, USA), Phospho-ERK1/2(Thr202/Tyr204) 1:1000 (9101S; Cell Signaling Technologies Inc.), and GAPDH 1:2000 (5174; Cell Signaling Technologies Inc) were used. The membrane was washed and incubated in horseradish peroxidase-conjugated secondary antibodies (GE Healthcare, Buckinghamshire, UK). Antibody-bond protein bands were assayed using a chemiluminescent luminol enhancer solution (Cyanagen, Bologna, Italy).

TA muscles were dissected, minced, and homogenized in lysis buffer (50 Mm Tris HCl pH 7.4, 1 mM EDTA, 150 Mm NaCl, 1% Triton) supplemented with protease and phosphatase inhibitors. Proteins (30–50 μg) were separated by SDS-PAGE and transferred to PVDF membrane (Invitrogen). Unspecific binding was blocked with 5% not fat dry milk in TBST buffer (20 mM Tris-HCl pH 7.6, 137 mM NaCl, 0.5% Tween 20) then membranes were incubated overnight at 4 °C, with primary antibody diluted in 5% BSA (Sigma) in TBST. After washing in TBST, membranes were incubated with secondary antibodies HRP-conjugate (Bio-Rad 170-6515 or 170-6516) and signals were detected by luminol-enhancer solution (Cyanagen). Images were acquired using films or ChemiDoc MP imaging system (Bio-Rad). Densitometric analyses were performed by measuring band intensity for each sample using ImageJ software. The following primary antibodies were used: Gapdh (Santa Cruz sc-32,233), LC3b (Cell Signaling 2775), p62 (Sigma P0067), Gp91phox (BD Transduction Laboratories), MF20 (Developmental Studies Hybridoma Bank).