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Cell signaling assays For analysis of human LepR mutants, HEK-293T cells were plated in six-well culture dishes coated with fibronectin (Millipore) at 0.7 × 10 6 cells per well in DMEM (Dulbecco’s Modified Eagle Medium) supplemented with 10% v/v fetal bovine serum, penicillin-streptomycin, 1 mM sodium pyruvate, 10 nM HEPES and 2 mM GlutaMAX TM (Gibco)

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Next, the protein concentration was quantified using the BCA Protein Concentration Assay Kit (#P0012, Beyotime)

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