Nuclear and cytoplasmic extraction kit

The first trifoliate leaf of GmFT3b-overexpressing seedlings was harvested and conserved under −80 °C. The total soluble proteins were extracted using the plant protein extraction protocol as described previously [50 (link)]. The total nuclear proteins were extracted using the nuclear and cytoplasmic extraction kit (CWBIO, Beijing, China). After protein denaturation, samples were separated by 10% SDS-PAGE and then analyzed by immunoblot using 1:3000-fold dilution anti-GFP mouse monoclonal antibody. In addition, the PVDF membrane was incubated with the One Step Western Kit HRP (CWBIO, Beijing, China). Finally, the immunized proteins were imaged under the Amersham Imager 600 (GE Healthcare, Little Chalfont, Buckinghamshire, UK) machine.

Nuclear and cytosolic proteins were separated using a nuclear and cytoplasmic extraction kit (CWBIO, Beijing, China). HUVECs were briefly washed with cold PBS, followed by dissociation in cold buffer A containing 1% phosphatase inhibitor and protease inhibitor cocktail. The supernatant containing cytosolic proteins was collected after centrifugation at 12,000 rpm for 15 min at a low temperature. Then, the nucleoprotein lysate containing protease inhibitor cocktail was added to the remaining sediment for further cleavage, and the samples were placed on ice for half an hour. Subsequently, the supernatant containing nuclear proteins was transferred to the corresponding test tube after centrifugation at 12,000 rpm for 15 min. The BCA assay kit was used to detect protein concentration according to the provided procedure. Then, the loading buffer was added according to a quarter of the amount of protein solution, followed by boiling for 5 min. The cytosolic and nuclear proteins were preserved at −80 °C until use.