Sc 13036

Freshly isolated myocytes were plated onto laminin-coated 8-well glass coverslips and fixed with 4% paraformaldehyde. Cells were then permeabilized with 50 µg/mL saponin (15 min), blocked with 10% goat serum and 10% BSA for one hour and incubated overnight at 4 °C with primary antibodies against NFATc4 (Santa Cruz sc-13036; 1:100 dilution) or HDAC4 (Santa Cruz sc-11418; 1:100 dilution). Myocytes were then washed (6x) with PBS containing 1% BSA and incubated with Alexa Fluor 488 conjugated anti-rabbit secondary antibody (ThermoFisher A11034; 1:50 dilution) for 2 h at room temperature. Cells were washed again in PBS and imaged with a laser scanning confocal microscope. Signal intensities were analyzed in Image J.

The Duolink-PLA assay (Olink, Uppsala, Sweden) was performed according to manufacturer’s protocol with minor modifications regarding antibody concentration. Antibodies for NFATc4 (sc-13036, Santa Cruz, Dallas, TX, USA), NFATc1 (sc-1149, Santa Cruz), GRK2 (sc-18409, Santa Cruz) and PI3Kγ (sc-7177, Santa Cruz) were added at a concentration of 6.7 μg/ml. The green fluorescence dots of each ligation, indicating protein-protein interaction, were detected using a Nikon laser scanning confocal microscope (Nikon C2+, Düsseldorf, Germany) and related to cell area. Other fluorescence assays were analyzed using an Olympus IX81 florescence microscope (Olympus, Hamburg, Germany). Fluorescence microscope images and intensity signals (green spots) were counted using ImageJ software (National Institute of Health, Bethesda, MD, USA).