Anti phosphotyrosine

Anti‐p130cas, anti‐myosin light chain kinase (MLC), anti‐phospho p130Cas (Y165), and anti‐phosphopaxillin (Y118) antibodies were from Cell Signaling Tech., Danvers, MA, USA. Anti‐paxillin antibody was from ECM Biosciences LLC, Versailles, KY, USA, Anti‐FAK (focal adhesion kinase), anti‐vinculin, anti‐phosphotyrosine, and anti‐phospho MLC (Thr18/Ser19 serine) antibodies were from Santa Cruz Biotechnology, Dallas, TX, USA. Anti‐phospho‐vinculin (Y1065) was from abcam, Cambridge, MA, USA. Alexa Fluor 546‐conjugated secondary antibody, Alexa Fluor 488‐conjugated phalloıdin, Fluorescein isothiocyante (FITC)‐labeled dextran and ROCK inhibitor y27632 were purchased from Thermo Fisher Scientific, Waltham, MA, USA. RhoA inhibitor I, (purified C3 Transferase covalently linked to a proprietary cell penetrating moiety), was purchased from Cytoskeleton Inc., Denver, CO, USA. HMEC‐1 (human microvascular endothelial cell‐1) 10 was obtained from the Centers for Disease Control and Prevention (Atlanta, GA) and cultured in vascular cell basal medium supplemented with endothelial cell growth factor kit (Invitrogen, Waltham, MA, USA). Dasatinib was obtained from Selleck Chem., Houston, TX, USA.

The isolated ventricular myocytes were lysed in lysis buffer containing (in mM) 150 NaCl, 20 HEPES, 1 EDTA, and 0.1 PMSF, as well as 1% Triton X-100 (in μg ml⁻¹), at pH 7.2, and the lysates were centrifuged at 13,000 g for 10 min. The proteins were then immunoprecipitated with rabbit IgG (Sigma), mouse anti-SERCA (Affinity BioReagents), mouse anti-α-actin (Sigma) or mouse anti-PLB antibodies (Affinity BioReagents) (1:100 dilution). The immune complexes were subsequently collected by adding protein A or G beads (1/10 volume, Sigma), fractionated by 10% SDS-PAGE, and transferred to polyvinylidene difluoride membranes. The blots were incubated with anti-phosphotyrosine (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-α-actin, anti-PLB antibodies, anti-phospho-Ser,^{16 (link)} or anti-phospho-Thr^{17 (link)} PLB antibodies (Badrilla, Leeds, UK) and subsequently incubated with goat anti-mouse or anti-rabbit alkaline phosphatase-conjugated secondary antibodies (Santa Cruz Biotechnology). The proteins were visualized using an enhanced chemiluminescence system (Intron, Seongnam, Republic of Korea) and an LAS 3000 imaging system (Fuji, Tokyo, Japan). To detect the total or tyrosine-phosphorylated α-actin in SR vesicles, we removed the IgG heavy chain band using ImmunoCruzTM IP/WB Optima E (Santa Cruz Biotechnology, Inc.) according to the manufacturer’s instructions.

Whole-cell lysates were resolved by SDS-PAGE (40 μg of protein was loaded into each well) using 10% acrylamide mini-gels, followed by electrophoretic transfer to PVDF membranes (Immobilon-P MILLIPORE) for 2 h. Membranes were blocked with 5% fat-free milk in PBS for 2 h and incubated with primary antibodies overnight. The detection step was performed with peroxidase-coupled anti-mouse IgG and anti-rabbit IgG (BioLegend, 1 : 2000) and anti-goat IgG (Santa Cruz Biotechnology) for 2 h. The primary antibodies included antiphosphotyrosine (Santa Cruz Biotechnology) and β-Actin (BioLegend). All primary antibodies were diluted 1 : 500 in 1% fat-free milk in PBS. Blots were developed with the ECL detection system according to the manufacturer's instructions (Amersham). Blots are representative of two separate experiments.