CoIP assay

Potential interaction between X. tropicalis Fosl1 (xFosl1) and xJunB proteins was examined by CoIP assay in X. tropicalis embryos. CoIP was performed by using the Pierce™ Classic Magnetic IP/Co-IP Kit (#88804, Thermo Scientific). The Flag-tagged xFosl1 and HA-tagged xJunB plasmids were co-injected into the animal pole of X. tropicalis embryos (fertilized eggs) at one-cell stage, using microinjector (PV-820, WPI). Each embryo was injected with 2 nl solution containing both plasmids (100 pg per plasmid). On the next day, injected embryos were inspected, and abnormal ones were then removed. Proteins were extracted from the normally developed embryos 48 h after injection, by using ice-cold IP lysis/wash buffer supplemented with protease inhibitor. The mixture was incubated on ice for 5 min with periodic mixing and centrifuged at 13,000 × g for 10 min to gain the supernatant. Second, the supernatant (containing 8 μg protein) was incubated overnight with anti-Flag (Sigma, F1804) or anti-HA (CST, #3724) at 4 °C on a gentle shaker. Magnetic bead-conjugated mouse IgG (CST, #5873) or normal rabbit IgG (CST, #2729) was used as negative control. Third, 25 µl magnetic beads were washed by using 175 µl IP lysis/wash buffer gently and washed by using 1 ml IP lysis/wash buffer again after discarding the supernatant. Magnetic beads and IP sample were mixed gently and incubated at room temperature on slow rotation for 1 h. Beads were collected with magnetic stand, and the supernatant was removed. Five hundred microliters of IP lysis/wash buffer was added to the tube and gently mixed and the supernatant was discarded twice. Five hundred microliters of water was added to the tube and gently mixed. Finally, the antigen/antibody complex was eluted by elution buffer and analyzed by western blotting after denaturation at 100 °C for 7 min. To calculate the relative expression of xFosl1-Flag and xJunB-HA in embryos, β-actin serves as a reference for the sample loading. In addition, the interaction between Fosl1 and JunB during neonatal mouse heart regeneration was also determined by CoIP assay as described above using anti-Fosl1 (Abcam, ab252421) and anti-JunB (Abcam, ab128878) antibodies. All blots are derived from the same experiment and were processed in parallel. The uncropped blots are listed in Supplementary Fig. 11.