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Lysis buffer RIPA (Thermo Fisher Scientific) and N-PER (Thermo Fisher Scientific) were used to extract proteins from glioma cells and tissues, respectively. Then, 50 μg total protein per sample was separated using 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Thermo Fisher Scientific). The membranes were probed with primary anti-IRAK3 antibody (ab8116, Abcam, MA, USA), anti-MEK1 (phospho S298) antibody [EPR3338] (ab96379), anti-ERK1 + ERK2 antibody [ERK-7D8] (ab54230), anti-c-Fos (phospho T232) antibody (ab17933), and anti-GAPDH antibody [6C5] (ab8245, Abcam) as control. The number of binding proteins was measured using AlphaEaseFC software (Genetic Technologies, FL, USA).

Primary cortical neurons were isolated from E18.5 C57/B6 mouse embryos. Briefly, cortical tissues from the brains of the mouse embryos were digested with 0.05 % trypsin‐EDTA.Na₂ at 37°C for 12 min and dissociated cortical cells were plated at a density of 1 × 10⁵ on poly D‐lysine‐coated 24‐well plates in Planting Medium (DMEM‐H medium with 10 % [vol/vol] FBS, 1 % [vol/vol] GlutaMAX, and 1 % [vol/vol) penicillin–streptomycin). After 4 h, cells were cultured in Neuron Medium (Neurobasal medium containing 1/50 [vol/vol] B27 and 1 % [vol/vol] GlutaMAX) for another 14 days for screening and maturation in a humidified 5 % CO₂ incubator at 37°C.
To determine the signaling pathways of GPR30, cultured cortical neurons were treated with DMSO, GPR30 agonist (G‐1, Cayman Chemical Company, USA) (1 μM), or GPR30 antagonist (G‐15, Cayman Chemical Company, USA) (1 μM) for 1 h for 14 days. After the treatments, cultures were washed with cold PBS (pH 7.2), lysed in cold lysis buffer (N‐PER, Thermo Scientific, MA), and then harvested with a cell scraper, followed by centrifugation at 12,000 rpm for 10 min. Protein concentrations were determined using the BCA Assay (Pierce Biotechnology, IL).