Bl612a

Total protein was extracted from the hippocampus and prefrontal cortex tissue with RIPA protein lysate (P0013, Beyotime, China) added with protease inhibitor (BL612A, Biosharp, China) and phosphatase inhibitor (BL615A, Biosharp, China), and normalized after quantification by BCA kit (P0009, Beyotime, China). The denatured proteins were separated by SDS-PAGE and transferred to PVDF membranes (Sigmaaldrich, USA). After being closed with 5 % skim milk for 2 h, primary antibodies RAS (1:1000; 3965, Cell Signaling Technology, USA), RAF1 (1:2000; 26863-1-AP, proteintech, China), MEK1/2 (1:2000; AF6385, affinity, China), p-MEK1/2 (1:1000; AF8035, affinity, China), ERK1/2 (1:2000; A16686, abclonal, China), p-ERK1/2 (1:2000; AP0472, abclonal, China), and β-actin (1:50000; AC026, abclonal, China) were incubated at 4 °C overnight. The second antibody Goat Anti-Rabbit IgG (H + L) HRP (1:5000; S0001, affbiotech, China) was incubated at room temperature for 2 h. The ECL developer (17046, zen-bio, China) and protein imaging system (5200 Multi, Tanon, China) visually analyze the proteins. The relative expression of the protein was calculated using β-actin as a reference.

The cells were lysed in 10% sodium dodecyl-sulfate (SDS) containing 1% protease inhibitor (BL612A; Biosharp, Hefei, China), and the protein concentration was quantified via Bradford protein assay. A total of 10 µg of protein lysates from each sample was separated in 10% SDS–polyacrylamide gel electrophoresis (PAGE) and then transferred onto polyvinylidene fluoride membranes (IPVH00010; MilliporeSigma, Burling, MA, USA). After blocking was completed with 5% skim milk, the membranes were incubated overnight in a 4 ℃ refrigerator with the primary antibodies against SLC16A1 (20139-1-AP; Proteintech, Wuhan, China) and GAPDH (KM9002; Sungene Biotech, Tianjin, China). After being washed, the membranes were probed with anti-rabbit immunoglobin G (IgG) secondary antibody (ab205718; Abcam, Cambridge, UK) or anti-mouse IgG secondary antibody (ab6728; Abcam).