Ecl chemiluminescence

An equal amount of protein from each sample was separated by SDS-PAGE, transferred onto a PVDF membrane (NEN Life Science Products, Massachusetts, USA), recognized by rabbit anti-LC3 (microtubule-associated protein light chain 3) antibody (1:2000) (Sigma), rabbit anti-α-II spectrin antibody (1:1000, Santa Cruz), or mouse anti-β-actin antibody (1:5000) (Chemicon, CA, USA), followed by secondary antibody (goat anti-rabbit 1:5000 or goat anti-mouse 1:10,000); then visualized by ECL chemiluminescence (Perkin Elmer, Massachusetts, USA). The relative level of LC3-II to LC3-I depicted the level of autophagy [21 (link)]. The ratio of spectrin breakdown products with molecular weights of 145 or 150 kDa (SBDP 145/150) to spectrin served as an index of injury severity [22 (link)].

The descending aortas were used for Western blotting assay. In detail, the total protein was extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology, Inc., Haimen, China). Then the protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). After 2 h of blocking by Tris Buffered Saline with Tween 20 (TBST) buffer with 5% non-fat milk, the PVDF membrane was incubated overnight at 4°C with primary antibodies against phosphorylated (p)-PI3K p85 (Y458) + PI3 Kinase p55 (Y199), p-AKT (Ser473), p-mTOR (Ser2448), PI3K, AKT, mTOR, Beclin 1, P62 and microtubule-associated protein 1A/1B-light chain 3 II (LC3II) (diluted at 1:1000, Abcam or CST). Following that, the membrane was incubated with the secondary antibody for 1 h. The intensity of protein expression was detected by ECL chemiluminescence (PerkinElmer, Inc., Boston, MA, USA), with GAPDH as an internal normalization control.