Ab183097

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.

For immunohistochemistry, representative samples (n = 4, 2 males and 2 females from separate litters) from each group were blocked with 3% hydrogen peroxide and then washed 3 times in phosphate buffered saline and blocked in 1% goat serum or donkey serum with 1% bovine serum albumin. Sections were incubated with the following primary antibodies overnight at 4 degrees: nicotinic acetylcholine receptor α3 (AbCam Cambridge, MA, ab183097, 1:50), α7 (ab10096, 1:200), β2 (ab129276, 1:100), β4 (ab189174, 1:400). Then, sections were washed 3 times in phosphate buffered saline and incubated with HRP conjugated secondary antibody for 1-hour (ab6721, ab6885, 1:250) and diaminobenzidine (DAB) (Vector Laboratories, Bulingame, CA) chromogen was used according to manufacturer’s protocol to identify immunoreactive structures. Coronal suture and whole synchondroses including abutting trabecular bone were digitally isolated for direct comparison between control and nicotine exposed individuals (outlined in Fig. 2d). At least 3 sections 30 μm apart per individual per treatment for each target were analyzed using Image J Software and the IHC Profiler Open Source Plugin for automated scoring of percent positivity^{41 (link)}.