Verification of interaction between JDP2 and PDE4B

For the dual luciferase reporter assay, H9c2 cells (5x10⁵ cells/well) were seeded (1x10⁴ cells/well) into 96-well plates. Subsequently, H9c2 cells were co-transfected with PDE4B-WT and PDE4B-MUT, Oe-JDP2 or Oe-NC (0.5 µg) and Renilla luciferase reporter vector (Promega Corporation) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) for 48 h at 37˚C. At 48 h post-transfection, firefly luciferase activities was measured using a Dual Luciferase Reporter Assay system (Promega Corporation) and normalized to Renilla luciferase activities.

For the chromatin immunoprecipitation (ChIP) assay, ultrasound-treated samples were centrifuged at 12,000-14,000 x g for 5 min at 4˚C. A total of 300 µl SDS lysis buffer (Active Motif, Inc.) was then used to lyse the cells, which were subsequently sonicated at 150 Hz and sheared with four sets of 10 sec pulses on wet ice using a high intensity ultrasonic processor. ChIP Dilution Buffer containing 1 mM PMSF was prepared using the ChIP Assay kit (Beyotime Institute of Biotechnology) and added to part of the samples to serve as the input. A total of 40 µl protein A/G agarose beads (Santa Cruz Biotechnology, Inc.) was added to the rest of the samples. Following centrifugation at 16,000 x g for 10 min at 4˚C, supernatant (100 µl) was incubated with 5 µg anti-IgG (cat. no. sc-2025; Santa Cruz Biotechnology, Inc.) or anti-JDP2 (cat. no. sc-517133; Santa Cruz Biotechnology, Inc.) primary antibodies and protein A/G agarose at 4˚C overnight. The precipitated chromatin was purified by phenol/chloroform/isoamyl extraction and analyzed via RT-qPCR according to the aforementioned protocol.