Rabbit anti creb

A total of 50 μg of cell lysate were resolved using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and blotted electrophoretically to polyvinylidene fluoride membranes. The membranes were then incubated with primary antibodies, and immunoreactivity was detected using horseradish-conjugated secondary antibody and visualized using enhanced chemiluminescence. The following primary antibodies were used: Rabbit anti-GLUT1 (1:500 Santa Cruz, CA, USA), mouse and rabbit anti-GLUT3 (1:500 Santa Cruz, 1:1000 Abcam), rabbit anti-pCREB (1:1000 Abcam), rabbit anti-CREB (1:1000 Abcam), rabbit anti-N-cadherin (1:1000 Abcam), rabbit anti-E-cadherin (1:500 Santa Cruz), mouse anti-HK2 (1:500 Santa Cruz), mouse anti-Ki-67 (1:500 Santa Cruz), and mouse anti-β-actin (1:1000, Abcam). Secondary antibodies (a donkey anti-goat IgG antibody (1:10,000, Jackson lab, PA, USA), a goat anti-mouse IgG antibody (1:10,000, Jackson lab), and a goat anti-rabbit IgG antibody (1:10,000, Jackson lab)) were coupled to horseradish peroxidase (HRP) for 1 h at room temperature.

At the same time points following the procedures [32 (link)], proteins were extracted from the MCA-supplied brain regions and loaded onto SDS-polyacrylamide gel for electrophoresis. Gel transfer to a PVDF membrane was performed under 200 V for 1 h. Membranes were blocked with 5% skimmed milk. Membranes were incubated with primary antibodies (1:1000, rabbit anti-BDNF, rabbit anti-NGF, rabbit anti-PSD-95, rabbit anti-SYN, mouse anti-Tau, rabbit anti-GAP43, rabbit anti-VEGF, rabbit anti-Ang-1, rabbit anti-Ang-2, rabbit anti-TrkB and rabbit anti-CREB, Abcam, MA, USA; 1:500, rabbit anti-HIF-1α, Santa Cruz Biotechnology, Inc. CA, USA) for 24 h at 4 °C. The secondary antibody was goat anti-rabbit IgG-HRP (Santa Cruz), which was incubated for 1 h at room temperature for all primary antibodies. Western blot images for each of the antibodies were analyzed using an image analysis program (ImageJ 1.42, National Institutes of Health, Bethesda, MD, USA) to quantify protein expression in terms of relative image density.

Cells were rinsed twice with PBS, lysed in RIPA lysis buffer (Beyotime, China, CAT#P0013B) supplemented with a protease inhibitor (PMSF, Beyotime, China, CAT#ST506) and Phosphatase inhibitor cocktail A (Beyotime, China, CAT#P1082), and protein concentration was measured using the BCA reagent (Beyotime, China, CAT#P0012). Electrophoresis was conducted by 10% SDS-polyacrylamide gel and transferred proteins to PVDF membranes (Millipore, Cat# IPVH00010). The following primary antibodies were used for incubation overnight at 4 °C: 1:10,000 mouse anti-beta-Tubulin (Proteintech, China Cat#66240-1-Ig), 1:1000 rabbit anti-PKA (Cell Signaling, USA Cat#5842 T), 1:5000 rabbit anti-pCREB (Abcam, UK Cat# ab32096), and 1:1000 rabbit anti-CREB (Abcam, UK Cat# ab32515). All membranes were incubated with HRP-conjugated Affinipure Goat Anti-Mouse IgG (H+L) (Proteintech, China Cat#SA00001-1) or HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) (Proteintech, China Cat#SA00001-2) for 1 h. Immunoreactive bands were detected using an enhanced chemiluminescence (ECL) kit (Advansta, USA Cat#K-12045-D10) and quantified with a gel-image analyzing system (Fusion Optix, USA).