Western Blot Analysis of Neuronal Receptors

Protein lysates from the VMH, LHA and BAT were subjected to SDS-PAGE, electrotransferred to polyvinylidene difluoride membranes (PVDF; Merck Millipore; Billerica, MA, USA) with a semidry blotter and probed with antibodies against UCP1 (1:10,000; ab10983), MOR (1:1000; ab17934), β2-nAChR (1:5000; ab41174), β4-nAChR (1:1000; ab129276), α3-nAChR (1:1000; ab183097), α4-nAChR (1:1000; ab41172), α7-nAChR (1:1000; ab216485) (Abcam; Cambridge, UK); β-actin (1:5000; A5316), α-tubulin (1:5000; T5168), KOR (1:1000; SAB2501442), DOR (1:1000; SAB4502042) (Sigma; St Louis, MO, USA); AMPKα1 (1:1000; 07–350), AMPKα2 (1:1000; 07–363) (Millipore; Billerica, MA, USA), pAMPKα-Thr¹⁷² (1:1000; 2535S) (Cell Signaling; Danvers; MA, USA)^{3 (link),48 (link)–50 (link),54 (link)}, Autoradiographic films (Fujifilm, Tokyo, Japan) were scanned and the bands signal was quantified by densitometry using ImageJ-1.44 software (NIH; Bethesda, MD, USA)^{3 (link),48 (link)–50 (link),54 (link)}. Values were expressed in relation to β-actin (hypothalamus) or α-tubulin (BAT). Representative images for all proteins are shown; all the bands for each picture come always from the same gel, although they may be spliced for clarity. Uncropped and unprocessed scans of the showed blots are supplied in the Source Data file.