Ab2157

Total protein was extracted from cells using radioimmunoprecipitation assay buffer (Beyotime, China) and the protein was separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as previously described [33 (link)]. The antibodies used were as follows: anti-AKT3 (Abcam, Cambridge, MA, USA; ab2157); anti-p-AKT (Abcam; ab8805); anti-P-AKT (Abcam; ab8805; ab192623); and anti-GAPDH (Abcam; ab9485). Original Western Blot images were provided in Supplementary file 1.

Protein homogenates were prepared from BAT axillary tissue as described (Hindle and Martin, 2014 (link)) from
n = 3 samples for each IBA, LT, EAr, and SpW groups.
Western blots were used to measure PNPLA2 (1:1000, rabbit pAb #2138, Cell
Signaling Technology, Danvers, MA) and β-tubulin (1:1000, goat pAb #ab21-57,
Abcam, Cambridge, MA), both detected using IRdye-conjugated secondary antibodies
(1:20,000 IRDye 800CW anti-rabbit and 1:10,000 IRDye 680LT anti-goat; Li-Cor,
Lincoln, NE). Proteins were imaged (Odyssey near-infrared imaging system, Li-Cor)
and analyzed with ImageQuant TL software (GE Healthcare). To correct for
inconsistencies in protein loading, each PNPLA2 band was normalized to the
β-tubulin band within the same lane. Changes in PNPLA2 abundance among the
sample groups were assessed by a one-way ANOVA (α = 0.05).