Anti phospho creb

NMDAR, p-CREB, and CD11b expression in the striatum was evaluated by immunofluorescence. Briefly, the brain tissue was frozen and the mid-part of the striatum was cut into 20 μm slices in the coronal plane. The slices were washed three-times in PBS (pH 7.4) for 5 min each time, blocked with 10% goat serum, and incubated with the following primary antibodies overnight at 4 °C: anti-phospho-CREB (1:1000, Abcam), NMDAR (1:1000, Abcam), and CD11b (1:1000, Abcam). The slices were then incubated with goat anti-rabbit secondary antibody (1:500, Beyotime) for 2 h in the darkness at room temperature, and washed three-times in PBS for 10 min each time. Images were captured using a fluorescence microscope (IX71; Olympus Corporation, Tokyo, Japan).

Total proteins were extracted from cells using PhosphoSafe Extraction Reagent (Novagen). Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to 0.2-μm PVDF membranes. The membranes were blocked in 5% BSA in TBS-Tween 20 (TBS-T) for 2 h and incubated overnight at 4°C with the following primary antibodies: anti-ADCY1 (Santa Cruz), anti-phospho-MEK1/2 (Cell Signaling Technology), anti-total-MEK1/2 (Cell Signaling Technology), anti-phospho-ERK1/2 (Cell Signaling Technology), anti-total-ERK1/2 (Cell Signaling Technology), anti-phospho-CREB (Abcam), anti-total-CREB (Proteintech), anti-MITF (Abcam), anti-N-cadherin (Proteintech), anti-E-cadherin (Proteintech), anti-vimentin (Proteintech), and anti-slug (Proteintech). After washing with TBS-T for 3 times, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (MBL) for 1 h. Bands were visualized with ECL Select^TM Western Blotting Detection Reagent (GE Healthcare), and bands intensities were quantified using ImageJ software.