Mouse anti parkin

Total protein was extracted from tissues by RIPA, and the concentration was measured with BCA Protein Quantitative kit. After denaturing by boiling for 5–10 min, the proteins were loaded, separated by SDS-PAGE gel electrophoresis and transferred onto nitrocellulose filter (NC) membranes. After blocked with 5% skim milk or 5% Bovine albumin (BSA), the NC membranes were incubated with the following antibodies at 4°C overnight: rabbit anti-SIK2 (Thermo Fisher; 1:1000); rabbit anti-HIF-1α (Bioss; 1:1000); rabbit anti-mTOR (Cell Signaling; 1:1000); rabbit anti-p-mTOR (Cell Signaling; 1:1000); rabbit anti-PINK1(Abcam; 1:1000); mouse anti-Parkin (Abcam; 1:1000). After rinsed with Tris-buffered saline with Tween (TBST), these membranes were incubated with goat anti-rabbit or anti-mouse IgG-HRP (Biosharp; 1:10000), and exposed with ECL reagent (Biosharp). β-actin was used as the endogenous control.

Alpha-linolenic acid (ALA), Human Beta Amyloid 1-42 (Aβ_1-42), All-trans retinoic acid (RA), Insulin, Human Insulin ELISA Kit, Human IGF-I ELISA Kit, 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (MTT), bovine serum albumin (BSA), and acridine orange (AO) were purchased from Sigma Aldrich, Saint Louis, MO, USA. Insulin Degrading Enzyme (IDE) was purchased from Abcam Inc., USA. Antibodies to the following targets were used: rabbit anti-TOMM20 antibody (Abcam Inc.), mouse anti-PARKIN (Abcam Inc.), rabbit anti-Synaptophysin (GeneTex, Inc., Alton Pkwy Irvine, CA, USA), mouse anti-β3-Tubulin (TUJ 1; Santa Cruz Biotechnology, Dallas, CO, USA), horseradish peroxidase- (HRP-) conjugated goat anti-rabbit IgG antibodies (Abcam Inc.), HRP-conjugated goat anti-mouse IgG antibodies (Abcam Inc.), Alexa Fluor™488-labeled chicken anti-mouse IgG (Thermo Fisher Scientific, Waltham, MA, USA), Alexa Fluor™568-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific).