Peroxidase conjugated affinipure goat anti mouse

D-Glucose and insulin were purchased from Sigma. Protease inhibitors were purchased from Merck. The following antibodies was purchased from Cell Signaling Technologies: Monoclonal rabbit anti-phospho-insulin receptor β (Tyr1150/1151, #3024), monoclonal rabbit anti-insulin receptor β (#3025, dilution used 1:1000), polyclonal rabbit anti-phospho-PKB (Ser473, #9271), monoclonal rabbit anti-PKB (pan, #4685), monoclonal rabbit anti-phospho-GSK3β (Ser9) (#5538), monoclonal mouse anti-GSK3β (#9832), and monoclonal mouse anti-phospho-PRAS40 (Thr246 (#13175S), all diluted at 1:1000. A monoclonal mouse anti-Actin was obtained from MP Biomedicals (#69100) and used in 1:10000 dilution. Secondary antibodies were purchased from Jackson Immuno Research, Peroxidase conjugated affiniPure goat anti mouse (#115-035-003) and Peroxidase conjugated affiniPure goat anti rabbit (#111-035-003) both were used in 1:1000 dilution.

Samples were loaded and separated by 6% SDS-polyacrylamide gel electrophoresis, and protein bands were subsequently transferred onto nitrocellulose membranes (Bio-Rad). Membranes were blocked with a 5% milk solution and then incubated overnight with antibodies at 4 °C. Immunoblots were incubated with secondary antibodies and visualized using Amersham Biosciences ECL Western blotting detection reagent (GE Healthcare Life Sciences). α-Tubulin was used as a loading control. After observing no intracellular calreticulin or α-tubulin with biotinylation (elution) blots (Fig. 4A), all subsequent elution blots are depicted without controls, as presented previously (83 (link)). Antibodies used were as follows: anti-TRPM7 (NeuroMab, clone N74/25, monoclonal), anti-α-tubulin (Sigma, monoclonal), anti-calreticulin (Stressgen, polyclonal), peroxidase-conjugated AffiniPure goat anti-rabbit (Jackson ImmunoResearch Laboratories, polyclonal), and peroxidase-conjugated AffiniPure goat anti-mouse (Jackson ImmunoResearch Laboratories, polyclonal).

Protease inhibitor cocktail (cat # P8340), sodium orthovanadate (cat # S6508) and dithiothreitol (cat # D9779) were purchased from Sigma Aldrich (St. Louis, MO, USA). Microcystin-LR (cat # 475815) was obtained from Calbiochem (La Jolla, CA, USA). Polyclonal chromogranin antibody (cat # ab12242) was purchased from Abcam (Cambridge, MA, USA). Monoclonal anti-drebrin antibody (D029-3) was purchased from MBL international corporation (Woburn, MA, USA). Polyclonal Rab3A antibody (15029-1-AP) was purchased from ProteinTech Group Inc. (Chicago, IL, USA). Rabbit polyclonal antibody against Gap-43 was obtained from Millipore (Temecula, CA, USA). Anti-flag tag antibody (M2; F3165) was purchased from Sigma Aldrich (St. Louis, MO, USA). Polyclonal Aβ oligomer antibody, clone A11 (cat# AHB0052) was obtained from Life Technologies (Grand Island, NY, USA). Mouse monoclonal antibody against beta-actin (cat # A00702) was purchased from Genscript USA Inc. (Piscataway, NJ, USA). Secondary antibodies such as peroxidase-conjugated AffiniPure goat anti-mouse (Code # 115-035-146) and ant-rabbit (code # 111-035-144) IgGs were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).