Autoradiographic films

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.

Protein lysates were subjected to SDS-PAGE, electrotransferred to a polyvinylidene difluoride membranes (PVDF; Merck Millipore; Billerica, MA, USA) and subsequently incubated with the following antibodies: ATF6α (90 kDa) (1:1000, sc-166659), CHOP (GADD153) (26 kDa) (1:1000, sc-7351), peIF2α (Ser52) (36 kDa) (1:1000, sc-12412), iNOS (1300 kDa) (1:1000, sc-7271) (Santa Cruz Biotechnology; Santa Cruz, CA, USA) and β-actin (42 kDa) (1:5000; A5316) (Sigma; St. Louis, MO, USA) after incubating the membranes with 3% BSA (β-actin, ATF6α), 5% BSA (peIF2α) or 5% skim milk (CHOP, iNOS) blocking buffer. Specific antigen–antibody bindings were detected using horseradish-peroxidase conjugated secondary antibodies (Dako Denmark; Glostrup, Denmark) and an enhanced chemiluminescence detection method, according to the manufacturer’s instructions (Pierce ECL Western Blotting Substrate; Thermo Scientific, Waltham, MA, USA) as described previously [127 (link),128 (link)]. Autoradiographic films (Fujifilm; Tokyo, Japan) were scanned and the band’s signal was quantified by densitometry using ImageJ-1.53 software (National Institutes of Health, Bethesda, MD, USA). Values were expressed relative to β-actin.

Total protein was obtained from frozen tissues through phenol extraction followed by methanol/ammonium acetate precipitation, as previously described (Hurkman and Tanaka, 1986 (link)). Peroxisomal and immunoprecipitated protein were obtained from the phenolic phase resulting from Tri-reagent RNA extraction, through precipitation in acetone, according to manufacturer’s instructions. Proteins were resolved by SDS-PAGE and electro-blotted onto Immobilion-P membrane (Millipore), which were then incubated with the appropriate antibody (@HA: Sigma–Aldrich ref. H6533; @HPR: Agrisera ref. AS11 1797; @P15: Incarbone et al., 2017 (link); @P19: kindly provided by K. Bouarab; @AGO2: Garcia et al., 2012 (link); @AGO1: Qi et al., 2005 (link)). After incubation with secondary antibody, membranes were revealed with Roche Lumilight Plus substrate (ref. 1201519600) and autoradiographic films (Fujifilm).