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NIH3T3 cells and hypothalamic fragments were homogenized with lysis buffer (mammalian cell protein extract reagent [M-PER], #78501; tissue protein extraction reagent [T-PER], #78510; Pierce Chemical, USA) containing protease inhibitor cocktail (Roche, Switzerland). Briefly, protein concentration was measured by the Bradford assay (#5000006; Bio-Rad, USA), and 20 μg of proteins from each sample were separated by SDS-PAGE and transferred onto PVDF membranes by electrophoretic transfer. The membrane was blocked with 5% non-fat skim milk in TBS-Tween and incubated with antibodies against NELL2 (1:1,000, #sc-390137; Santa Cruz Biotechnology, USA), pERK (1:1,000, #9101; Cell Signaling Technology, USA), ERK (1:1,000, #sc-153; Santa Cruz Biotechnology), or cFos (1:1,000, #sc-7202; Santa Cruz Biotechnology). The membrane was incubated with HRP-conjugated mouse secondary antibody (1:3,000, #7076; Cell Signaling Technology) or HRP-conjugated rabbit secondary antibody (1:3,000, #7074; Cell Signaling Technology), and the immunoreactive signals were detected by chemiluminescent detection reagent (#34095, Thermo Fisher Scientific, USA). Protein density was normalized using β-tubulin (1:3,000, #sc-5274; Santa Cruz Biotechnology) or β-actin (1:4,000, #A5441; Sigma-Aldrich), and Image J software was used to analyze data.