Cell mitochondrial isolation kit

Cytoplasmic and nuclear proteins were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo) according to the manufacturer’s instructions. In brief, H9C2 cells were harvested with trypsin-EDTA and lysed in CER. After centrifuging at 15,000 rpm for 5 min, the supernatant was collected as a cytosolic fraction. The remaining pellet was suspended in NER, and the supernatant was collected as nuclear fraction.
Mitochondria were isolated by cell mitochondrial isolation kit (Beyotime). In brief, after lysing and centrifuging at first time, the cell-debris pellet in the collection tube was used to extract nuclear protein, the supernatant was transferred to a new microcentrifuge tube and centrifuged at 16,000 g for 10 min, the remaining pellet was suspended with lysis buffer as mitochondrial protein. Nuclear protein was extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo).

PP7 was purchased from Chengdu Must Bio-Technology Co., Ltd. (Chengdu, China), and the purity of PPI was ≥98%. N-acetyl-L-cysteine (NAC) was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Insulin-like growth factor 1 (IGF-1) and epidermal growth factor (EGF) were purchased from PeproTech (Rocky Hill, USA). 3-methyladenine (3-MA, S2767) and bafilomycin A1 (Baf-A1, S1413) were from Selleck Chemicals (Houston, TX, USA). TMZ was supplied by Tasly Pharmaceutical Co., Ltd. (Tianjin, China). The Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologie (Kumamoto, Japan). Hoechst33342/PI kit, cell mitochondrial isolation kit, and dihydroethidium were supplied by Beyotime Institute of Biotechnology (Haimen, China).

For Western blot, embryos at the indicated developmental stages were deyolked and homogenized in lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 10% glycerol, and 0.1% Triton X-100) as previously reported [62 (link)]. Proteins from cell lines in lysis buffer (20 mM Tris, pH 7.4, 137 mM NaCl, 2 mM EDTA, 10% glycerol, 1% NP-40 (NEW 728601), and protease inhibitor cocktail (Roche, 04693132001) were stored on ice for 40 min as previously described. For immunoprecipitation, the cells were lysed for 40 min on ice in lysis buffer containing protease inhibitors (Roche Applied Science, 04693132001). Cell lysates were incubated with the indicated antibodies and protein A/G-agarose beads (Invitrogen) overnight at 4 °C. The beads were extensively washed with lysis buffer and then dissolved in sample buffer containing 1% SDS for 5 min at 95 °C for western blot analysis. Zebrafish or cell line mitochondria were isolated using Tissue Mitochondrial Isolation Kit (beyotime) or Cell Mitochondrial Isolation Kit (beyotime), respectively, as instructed. The antibodies used for western blot were as follows: rabbit anti-TOM20 (Santa Cruz), mosue anti-GAPDH (Sigma), rabbit anti-COXIV (cell signaling), rabbit-anti-actin (Sigma), rabbit anti-TAMM41 (Sigma), rabbit-anti-PINK1 (cell signaling), rabbit-anti-SOD2 (cell signaling), mouse anti-GFP (Invitrogen), mouse anti-HA (Sigma).

Mitochondrial extraction was performed by Cell Mitochondrial Isolation Kit (Beyotime, Cat. No. C3601, China). NP cells were washed with PBS, digested with Trypsin-EDTA solution, centrifugated at room temperature for 5-10 minutes. After cells were collected, Cells were added with 1-2.5ml of mitochondrial separation reagent and place in an ice bath for 10-15 minutes. And then the suspension was transferred to an appropriately sized glass homogenizer for about 10-30 homogenates. The cell homogenate was centrifuged at 600g at 4℃ for 10 min. The supernatant was transferred carefully to another centrifuge tube and centrifuged at 11,000g for 4 min. The supernatant was removed carefully and the precipitate was the isolated mitochondria. The separated mitochondrial samples were lysed by RIPA containing PMSF and were used for Western blot.

Act V (purity > 98%) and Act D (purity > 98%) were obtained from Dr. Xie (Shandong University, Weihai, China), isolated from Streptomyces sp. [33 (link)]. The compounds were dissolved in dimethyl sulfoxide (DMSO). Antibodies against AKT, PI3K, Bcl-2, Bax cleaved caspase-9, and cleaved caspase-3 were purchased from Cell Signalling Technology (CST Inc., Beverly, MA, USA). Antibodies against p-AKT, cytochrome C, and PARP were purchased from WanLei Biotechnology (Shenyang, China). Antibodies against GAPDH and β-Actin were purchased from Beyotime Institute of Biotechnology (Shanghai, China). The Annexin V-FITC apoptotic detection kit, JC-1 (“5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide”) staining kit, LY294002 (PI3K inhibitor), DAPI staining solution, and cell mitochondrial isolation kit were purchased from Beyotime Institute of Biotechnology (Shanghai, China). The HTRFKinEASE-TK kit was purchased from Cisbio (Cisbio Bioassays Co., Codolet, France). The PI3K antibody in the kinase inhibition assay was obtained from Abcam, Inc. (Cambridge, MA, USA). All chemicals used in this study were commercial reagent grade products.

According to the experimental grouping, the treated BMSCs were washed with phosphate-buffered saline (PBS; HyClone, U.S.A.) two to three times; then the cells were digested with 1 ml EDTA at 37°C, and then centrifuged at 600×g/min for 5 min. According to the instructions provided in the Cell Mitochondrial Isolation Kit (Beyotime, China), 1.5 ml of the mitochondrial separation reagent was mixed with the protease inhibitor PMSF to resuspend the cells, placed in an ice bath for 15 min, and then the cell suspension was transferred into a glass homogenizer and ground 30 times. The cell homogenate was obtained and centrifuged at 600×g/min for 10 min at 4°C, the supernatant was transferred to another centrifuge tube, the supernatant was centrifuged at 11000×g/min for 10 min at 4°C, and the supernatant was removed. The precipitate consisted of mitochondria and was immediately used or resuspended in 1 ml of mitochondrial storage solution and stored at −80°C.

To evaluate whether TroBcl2 inhibits the release of cytochrome c from mitochondria into the cytoplasm, we examined changes in cytochrome c protein levels in mitochondria-free cytoplasm after TroBcl2 overexpression and knockdown in GPS cells. Briefly, GPS cells were transfected with pTroBcl2 (or pCN3), and siTroBcl2 (or siTroBcl2-C) according to the transfection procedure described above. 24 h after transfection, cells were stimulated by 2 μg/ml LPS. Non-transfected cells treated with LPS were as the control. Twenty-four hours after LPS stimulation, proteins were isolated from of mitochondria-free cytoplasm using a cell mitochondrial isolation kit (Beyotime, Shanghai, China) and then analyzed using western blotting with a mouse anti-TroCyt c polyclonal antibody (stored in our laboratory, 1:1,000 dilution) as the primary antibody and HRP-conjugated goat anti-mouse IgG (1:2,000 dilution) as the secondary antibody. An anti-β-actin mouse monoclonal antibody (Bioss, Beijing, China) was used as an internal reference. Immunoreactions were detected with a supersensitive ECL substrate (Biosharp, Anhui, China).