Hippocampal tissue was treated with radio immunoprecipitation assay (RIPA) buffer and phenylmethanesulfonyl fluoride (PMSF), homogenized into a uniform suspension and centrifuged at 4°C for 15 minutes. The extracted proteins were then quantified using the BCA kit; 20‐25 µg of proteins from each group was loaded onto a 12% SDS‐PAGE gel, and after electrophoresis, the proteins were separated and transferred onto PVDF membranes. Non‐phosphorylated proteins were blocked by 7% non‐fat milk at room temperature for 2 hours and washed with TBST. Next, the protein‐loading membranes were incubated overnight at 4°C with the following primary antibodies: anti‐GAPDH (1:1000), anti‐LC3B (1:2000), anti‐p62 (1:10 000), anti‐Parkin (1:1000), anti‐MFN2 (1:5000), anti‐DRP1 (1:1000) and anti‐PGC1α (1:1000). After being washed with TBST, the membranes were incubated with the corresponding secondary antibody for 1 hour, and protein expression was detected using an ECL chemiluminescence kit. Membranes were then scanned with a ChemiDoc XRS (Bio‐Rad), and the resulting bands were quantified using Image Lab software v3.0.1 (Bio‐Rad).

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.