P erk1 2

The prefrontal cortices of mice were analyzed via immunofluorescent assays. Coronal sections (5-μm thickness) were prepared and immersed in 4% PFA for 30 min, followed by three washes in TBST. After blocking non-specific binding via 1.5% BSA for 1 h, sections were then incubated with corresponding primary antibodies overnight at 4 °C, as follows: GFAP (1:50; Proteintech, Wuhan, Hubei, China) and P-ERK1/2 (1:100; Beyotime, Shanghai, China); GFAP and GLUT3 (1:100; Santa Cruz, Dallas, Texas, USA); NeuN (1:50; Proteintech, Wuhan, Hubei, China); P-ERK1/2; and GFAP and T-ERK1/2 (1:100; Santa Cruz, Dallas, Texas, USA). After washing three times with TBST, the secondary antibody mix with FITC-conjugated goat anti-mouse IgG (1:200; Bioss, Beijing, China) and Cy3-conjugated goat anti-rabbit IgG (1:200; Bioss, Beijing, China) was added to cover the tissue and was incubated at room temperature for 1 h in the dark. Sections were then incubated with DAPI at room temperature for 5 min. Fluorescent images were obtained, and image analysis was applied to quantify immunoreactive signals.

Protein samples were extracted with lysis buffer. 15 μg protein samples from each group were loaded onto the SDS polyacrylamide gel for electrophoresis and transferred to NC membranes (Millipore, Billerica, MA, USA). After blocking in 5% non-fat milk at room temperature for 2 h, primary antibodies: NPRA (1:1000; Santa Cruz Biotechnology), NPRC (1:1000; Santa Cruz Biotechnology), PGRMC1 (1:1000; Cell Signaling), PGRMC2 (1:1000), Bax (1:2000; Proteintech), Bcl-2 (1:2000; Proteintech), Caspase 8 (1:1000; Proteintech), Caspase 9 (1:1000; Proteintech), PCNA (1:2000; Proteintech), EGFR (1:500; Proteintech), p-EGFR (1:500; Cell Signaling), ERK 1/2 (1:500; Beyotime Biotechnology), p-ERK 1/2 (1:500; Beyotime Biotechnology), p-c-Fos (1:500; Cell Signaling), c-Fos (1:500; Cell Signaling), p-c-Jun (1:500; Cell Signaling), c-Jun (1:500; Cell Signaling) and GAPDH (1:2000; Proteintech) were incubated at 4 °C overnight. Next day, secondary antibodies (1:2000; Cell Signaling) were incubated for 45 min at room temperature. ECL detection system was used to visualize the bands. All experiments were repeated at least three separate times.