Rabbit anti app ctf

Brains were homogenized using Qiagen Tissue-Lyser II (28 Hz, 3 min, 3 times) in lysis buffer (Cell Signaling #9803) containing protease inhibitor cocktail (Roche #4693159001) and PhosSTOP (Roche #4906837001) (1 ml lysis buffer for 100 mg tissue). Lysates were then incubated on ice for 10 min followed by centrifugation at 18,660 g for 20 min at 4°. Supernatants were transferred to new tubes for protein concentration determination and loading sample preparation. Protein lysate samples were boiled at 95°C and SDS-PAGE was performed using standard BioRad reagents. For Western blotting, PDVF membranes were incubated overnight at 4°C with the following primary antibodies diluted blocking buffer (Rockland): Mouse anti APP A4 clone 22C11 (Millipore MAB348, 1:1,000), Mouse anti APP clone 6E10 (Biolegend 803001, 1:2,000), Rabbit anti APP CTF (Sigma A8717, 1:4,000), Mouse anti-β-Actin (Sigma A2228, 1:4,000). Membranes were then incubated with the appropriate fluorescently conjugated secondary antibody (1:10,000, Li-Cor) and imaged using a Li-Cor Odyssey CLx system.

Mouse rabbit anti‐APP (Novus, 1:10,000), rabbit anti‐APP‐CTF (Sigma‐Aldrich, 1:5,000), anti‐ROCK1 antibody (Abcam, 1:500), rabbit anti‐SP1 (Abcam, 1:1,000), rabbit anti‐SP6 (Sigma‐Aldrich, 1:1,000), mouse anti‐sAPPα (IBL, 1:50), mouse anti‐Aβ (Cell Signaling, 1:1,000), and rabbit anti‐BACE1 (Cell Signaling, 1:1,000) were used. Loading controls (GAPDH, Sigma‐Aldrich, 1:1,000) were used for Western blot standardization. Lipofectamine 2000 was sourced from Invitrogen. Y‐27632 was purchased from Abcam.

Mouse anti-ROCK1 antibody (Abcam, 1:500 dilution), rabbit anti-Beclin1 (Novus, 1:10,000), rabbit anti-APP-CTF (Sigma-Aldrich, 1:5000), anti-mouse sAPPα (IBL, 1:50), anti-mouse myc (Abcam, 1:1000), anti-rabbit LC3 (Cell Signaling, 1:100) and mouse anti-β-actin (Sigma-Aldrich, 1:1000) were used. Loading controls (β-actin) were used for Western blot standardization. Lipofectamine 2000 was sourced from Invitrogen. 3-methyladenine (3-MA) was purchased from Sigma Aldrich (St. Louis, MO, USA) and 3-MA was dissolved in double distilled water and titrated to 1 mM.