Ab24233

Whole cell lysates were obtained from the dissected hypothalami from WT controls and FMR1 knockout mice and after protein determination, the same amount of protein from each sample was resolved on SDS-PAGE, transferred on nitrocellulose membrane and probed for: GABAγ2 receptor subunit (1:1000, 14104-1-AP, Proteintech), VGAT (1:1000, 131 011, Synaptic Systems), MC4R (1:1000, ab24233, Abcam) or β-tubulin (1:1000, sc-9104, Santa Cruz Biotechnology) (Supplementary information). Bands were quantified using ChemiDoc imaging system (Bio-Rad, Hercules, CA).

Upon euthanasia, mice were perfused with 4% paraformaldehyde prior to brain harvesting. The brains were transitioned to a 10% sucrose, a 20% sucrose, and finally a 30% sucrose solution in PBS, waiting until the tissue sank prior to each transition. The brain was sectioned in 35-40µm slices and then stored in cryoprotectant (20% glycerol and 30% ethylene glycol in PBS) until immunohistochemistry was performed. To examine MC4R localization on Sim1 neurons, brain sections were labeled overnight with rabbit anti-MC4R (Abcam ab24233) and then exposed to a Donkey anti-rabbit IgG Alexa Fluor 488 secondary antibody (Invitrogen, A21206). TdTomato was used as a cre-reporter; its fluorescence was visible via confocal (Leica TCS SP5) without the aid of an additional antibody.