*4.4. Protein Assay for Muscle and Adipose Tissue*

Portions of the right gastrocnemius muscle, inguinal WAT, and interscapular BAT were processed in a tissue homogenizer (Omni International, Kennesaw, GA, USA). Protein concentration of tissue homogenate was assayed using a Pierce BCA Protein Assay Kit (Thermo Scientific, catalog 23227, Waltham, MA, USA). Uncoupling (UCP) protein content in muscle and adipose tissue homogenates were assayed. In addition, adenosine triphosphate (ATP) content of tissue homogenate was assessed by using an ATP Assay Kit which relies on the phosphorylation of glycerol that could be quantified by colorimetric or fluorometric methods (Abcam, catalog ab83355, Cambridge, UK). Protein concentration of phospho-JAK2 and total JAK2, as well as phospho-STAT5 and total STAT5, in muscle homogenates was measured (Supplemental Table S1).
