Ecl plus reagents

The cells were dissected in RIPA lysate buffer with protease phosphatase inhibitor and protease inhibitor. The total protein concentrations were determined by BCA kit (Applygen, China) to ensure equal sample loading. Protein contents were separated on SDS-poly-acrylamide gels (10%) and then transferred into a 0.45 μm polyvinylidene fluoride membrane (Millipore, United States) which were blocked with 5% skim milk-TBST (20 mM Tris HCl, pH 7.5, 500 mM NaCl, 0.1% Tween 20) for 1 h. The membranes were probed with the following antibodies: β-ACTIN (1:10,000), Drp1 (1:1,000), p-Drp1 (Ser616) (1:1,000), GAPDH (1:1,000), TH (1:1,000), overnight at 4°C, and then incubated with secondary antibody (1:2,000, Abclonal, China) for 2 at room temperature. The blots were visualized by incubating the membranes with ECL Plus reagents (Yeasen, China) and the images were recorded by LAS-4000 chemiluminescence system (GE Healthcare, United States). The blot densities were assessed by Gel-pro analyzer 4.0.