Nuclear separation kit

Mitochondria were isolated using a mitochondrial isolation kit according to the manufacturer's instructions (Cayman). Briefly, mitochondrial separation solution was added to fresh LV tissue, after which the tissue was centrifuged at 700 × g for 10 minutes. The supernatant was then collected and centrifuged at 6000 × g for 10 minutes. The supernatant was discarded, and the pellet was collected after washing twice to obtain the purified mitochondria.
The nuclei were obtained from LV tissue using a nuclear separation kit (Njjcbio). First, the remaining fresh LV tissue was lysed with the kit-provided lysis buffer, and the tissue homogenate was collected and centrifuged at 1000 × g for 10 minutes. The collected pellet contained the nuclei. Nuclei with high purity were obtained by washing the pellet twice with the kit washing solution.

The cytoplasm and nuclei in LV tissue and RAW264.7 macrophages were isolated using a nuclear separation kit (Njjcbio) according to a previously described protocol [19 (link)]. Briefly, homogenate was collected from LV tissue and cells that were lysed in lysis buffer and further centrifuged at 1000 × g for 10 minutes. The pellets in the bottoms of the tubes contained the nuclei, and the supernatant contained the cytoplasm.
After the cytoplasm, nuclei, LV tissue, and HL-1 cardiomyocytes were, respectively, lysed in RIPA lysis buffer containing 10% protease inhibitors and 10% phosphatase inhibitors, the total protein was obtained and quantitated using a BCA Protein Assay Kit. After separation by electrophoresis on 10% SDS polyacrylamide gels, the proteins were transferred to PVDF membranes. Then, the protein expression of IL-16 (Abcam), Bax, Bcl-2, cleaved caspase-3, caspase-3, and GAPDH (all three from GeneTex) in total LV tissue, the protein expression of p-p65 and GAPDH (both from Abcam) in the cytoplasm, the protein expression of p-p65 and PCNA (GeneTex) in the nuclei, and the protein expression of Bax, Bcl-2, cleaved caspase-3, caspase-3, and GAPDH in HL-1 cardiomyocytes were measured using primary antibodies as indicated in parentheses. After further incubation with the secondary antibodies, the target proteins were detected and analyzed.