For Western blot analysis of various proteins in EC/HPC extracts, 10 to 20 μg of total protein per lane was loaded on 4 to 12% bis-tris SDS–polyacrylamide gel electrophoresis (SDS-PAGE) gels (Invitrogen) and separated by SDS-PAGE using MES or Mops running buffers (Invitrogen). After blotting on nitrocellulose membrane (Amersham), the membranes were blocked in PBS-based blocking buffer (LICOR) for 1 hour at room temperature and then incubated in primary antibody solutions overnight at 4°C. Antibodies in blocking buffer were as follows: mouse anti-human tau Tau13 (1:1000, BioLegend), chicken anti-GFP (1:1000, Aves), rabbit-anti GFAP (1:1000, Abcam ab7260), rat-anti Hsc70 (1:1000, Abcam ab19136), rabbit-anti Hsp70 (1:1000, Abcam ab79852), mouse-anti CHOP (alternative name DDTR1, 1:1000, Abcam ab11419), rabbit-anti PSMD13 (1:1000, Abcam ab229812), rabbit-anti Lamp3 (1:1000, ab83659), rabbit-anti LC3B (1:1000, Abcam ab51520), rabbit-anti p62 (1:1000, Abcam ab109012), mouse anti–phospho-tau PHF1 (1:1000, provided by P. Davis), mouse anti–phospho-tau 12e8 (1:1000, Prothena), goat anti-PSD95 (1:1000, Abcam ab12093), mouse anti–synapsin 1 (1:1000, Millipore MABN894), H2a.X (1:500, Abcam), and mouse anti-actin (1:1000, Millipore MAB1501R). After three washes in 0.02% Tween 20/TBS, the membranes were incubated with secondary antibodies in blocking buffer: goat anti-mouse/rabbit/rat-680 and donkey anti-mouse/rabbit/chicken/goat-800 (each 1:3000, LICOR, Rockland) for 2 hours at room temperature. Protein bands were visualized using a LICOR infrared scanner at wavelengths of 680 and 800 nm.

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.