Anti rabaptin5

Mice were anesthetized with ketamine (50 g/kg)/xylazine (5 mg/kg) and brains were rapidly removed over ice. Brains of 12 (n=4) and 24 (n=5) month old mice were snap frozen and stored at −80°C before biochemical analyses. Entorhinal cortices were dissected out from fresh brains of 12 (n=4) and 24 (n=4) month old mice and stored at −80°C. Western blot immunolabeling was performed as previously described^{52 (link)}. Primary antibodies used were: anti-LAMP-2 (1:1000), anti-Rab4a (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA; Cat # SC-312), anti-Rab5a (1:1000), anti-Rab7 (1:1000; Sigma, St. Louis, MO; Cat # R8779), anti-Rabaptin5 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA; Cat # SC-6162), anti-LC3 (1:1000), anti-Cat D (1:1000), C1/6.1 (1:1000)^{48 (link)}, anti-mTOR and anti-AKT (phosphorylated and total protein) (1:1000, Cell Signaling Technology, Inc., Danvers, MA; Cat # 2983S and Cat # 9272S respectively), anti-α-synuclein (1:1000; Sigma, St. Louis, MO; Cat # S3062), and anti-β-tubulin (1:10000; Sigma, St. Louis, MO; Cat # T8535). Secondary antibodies used were: HRP conjugated anti-rabbit and mouse antibodies (1:5000; GE Healthcare, Pittsburgh, PA). The protein bands were scanned, optical density was calculated using the Image J, and the ratio of protein intensity to β-tubulin in the same lane was calculated. Western blot analyses were repeated 3 times.