Hrp conjugated anti mouse igg

The megakaryocytes from different treatments (0, 10, 50 and 100 ng/mL, 12 d) were transferred to a glass homogenizer and ground in an ice bath for 35 min. Then, the mitochondrial proteins were prepared using a mitochondrial protein extraction kit according to the manufacturer’s instruction (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). After being quantitatively analyzed using a BCA protein assay kit (Beyotime, China), the consistent amount of the protein sample from each group was submitted to Western blot assay. The primary antibody was the total OXPHOS antibody cocktail (1:1000, ab110411, Abcam, USA), and the second antibody was the HRP-conjugated anti-mouse IgG (1:500, EasyBio, China). The quality of this assay was reversely confirmed by stable expressions of the test biomarkers, including mitochondrial respiratory chain complex II, III and V.

After stimulation with different coatings of 5 nm and 50 nm AuNPs (5 μg/mL) for 24 h, mitochondria in harvested cells were first obtained with a commercial kit following the manufacturer's protocol (Nanjing Jiancheng Biological Product, Nanjing, China). The mitochondrial proteins were then extracted using RIPA buffer lysis (Solarbio, Beijing, China). Following centrifugation at 10,000 rpm and 4 °C for 30 min, the concentrations of protein in the lysate were quantified by the BCA method (Thermo Scientific, Waltham, MA, USA). Mitochondrial extracted proteins were resolved by SDS-PAGE, transferred onto nitrocellulose membranes, and detected as described previously [69 (link)]. The total OXPHOS antibody cocktail (1:1000, ab110411, Abcam, Cambridge, MA, USA) and HRP-conjugated anti-mouse IgG (1:500, EasyBio, Beijing, China) were used as the primary antibody and secondary antibody, respectively.