Hax 1

Cells were washed with PBS and fixed in 4% paraformaldehyde over night at 4°C. Immunofluorescent staining was performed by using Opal^TM 7-color manual IHC Kits (PerkinElmer, United States). The cells were pretreated with microwave in AR buffer for 15 min and then blocked in block solution for 10 min. The primary antibody was incubated at 37°C for 2 h. After the cells were washed with TBST, secondary antibodies were used for 15 min (HRP conjugated, PerkinElmer, United States). Opal fluorophore working solution was then incubated for 10 min. After microwave treatment in AR buffer, another staining cycle was performed as describe above. Hsp90, Akt1 and HAX-1 were detected by different primary antibody (Hsp90 from Proteintech, China, Akt1 from CST, United States, and HAX-1 from BD, United States) and stained by different Fluorescent dyes (Hsp90 stained with red fluorescence, Akt1 stained with green fluorescence and HAX-1 stained with blue fluorescence). Laser scanning confocal microscopy (LSCM, Nicon) was used for cell observation.

Pull-down assays were performed as previously described [25 (link),56 (link)]. Briefly, wild-type mouse cardiac homogenates were prepared in 10 mM NaPO₄ (pH 7.2), 2 mM EDTA, 10 mM NaN₃, 120 mM NaCl, and 1% NP-40, supplemented with protease inhibitors (Sigma-Aldrich, Munich, Germany). Equivalent amounts of recombinant GST-PLN-WT and GST-PLN-R14del recombinant proteins were mixed with 0.5 mg of mouse cardiac homogenates at 4 °C for 16 h. The beads were washed with 10 mM NaPO₄ (pH 7.2), 10 mM NaN₃, 120 mM NaCl, and 0.1% (v/v) Tween-20 and were subsequently analyzed by Western blot with the following primary antibodies: SERCA2, Hsp90 (Cell Signaling Technology, Leiden, The Netherlands), HAX-1 (BD Biosciences, Erembodegem, Belgium), PP1, GM (Santa Cruz Biotechnology, Heidelberg, Germany), Hsp20, I-1 (AbCam, Cambridge, UK), HRC (Sigma-Aldrich, Munich, Germany), and peroxidase-conjugated goat anti-rabbit (GE Healthcare Life Sciences, Buckinghamshire, UK) or anti-mouse (Sigma-Aldrich, Munich, Germany) secondary antibodies. Immunoreactive bands were detected using Pierce ECL Plus reagents (ThermoFisher Scientific, Waltham, MA, USA). Protein quantification was performed using ImageJ [57 (link)].