Gata4 g 4

The experiments were performed as described previously [16 (link),32 (link)]. H9C2 were treated with 10⁻⁷ M phenylephrine (PE, Sigma-Aldrich Corporation, St. Louis, MO, USA) for 24 h. In some experiments, cells were treated with the synthetic peptide TAT-RH, which only reproduce the RH domain of GRK5, as described previously [18 (link)] or transfected with plasmids encoding for GRK5-NT or its mutant in the amino-terminal calmodulin binding. At the end of the treatment, cells were lysed in RIPA/SDS buffer, and protein concentration was determined by using Pierce BCA assay kit (Thermo Fisher Scientific, Waltham, MA, USA) [33 (link),34 (link)]. Total extracts were electrophoresed by SDS/PAGE and transferred to nitrocellulose [35 (link)]. The antibodies anti-NFATc4 (B-2) (SC-271597), GATA-4 (G-4) (sc-25310), p-NFATc4 (80.S168/170) (sc-135770), p-GATA-4 (H-4) (sc-377543), β-actin (C-4) (sc-47778), and histone H3 (FL-136) (sc-10809) were from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Inc, Dallas, TX, USA). In some experiments, nuclear proteins were isolated from heart samples as previously described [16 (link)]. Densitometric analysis was performed using Image Quant 5.2 software (Molecular Dynamics Inc., Caesarea, Israel). Images are representative of at least three independent experiments quantified and corrected for appropriate loading control.

Cells were fixed in 4% paraformaldehyde for 20 minutes and blocked in blocking buffer (PBS containing 5% BSA and 0.2% Triton X-100). Cells were incubated in primary antibody solution at 4°C overnight, followed by Alexa Fluor 488 (Invitrogen, 1:1000) secondary antibodies for 1 h at 37°C. Nuclei were stained with Hoechst (Invitrogen, 1:10000). The primary antibodies were Gata4 (G4; Santa Cruz, 1:100), Myosin (MF-20; DSHB, 1:50) and Troponin T (1:1000, Abcam).