Cpt1b

Paraffin sections (4 mm) were immunostained with antibodies against adipose differentiation-related protein (ADRP) (Abcam), F4/80 (Serotec), a-smooth muscle actin (Abcam), PPARa (Abcam), CPT1b (Abcam), or phosphorylated (phospho-) Thr 172 AMPK (Cell Signaling Technology). After incubating with primary antibody overnight at 4 C, the membranes were incubated with peroxidase-conjugated secondary antibodies (ZSGB-BIO, Beijing, China), and the signal was developed using 3,3'-diaminobenzidine tetrahydrochloride (ZSGB-BIO). Sections were then examined under a light microscope and digitized with a high-resolution camera.

20mg of frozen muscle tissue was freeze-dried and subsequently homogenized, separated and electroblotted as previosuly described (32) . The following primary antibody was purchased from Cell Signalling Technology (Danvers, MA, USA) and utilized as follows; phospho-specific ACC Ser79 (cat # 3661, conc. 1:1000 in 5% BSA). The following primary antibody was purchased from ABCAM (Cambridge, UK) and utilized as follows; CPT1B (cat # AB134135, conc. 1:1000 in 5% BSA). With regards to secondary antibodies for both targets, membranes were then incubated for 1 hr with Horseradish peroxidase-conjugated goat anti-rabbit (Cat # 2054, Santa Cruz,TX, USA) and utilized as follows: phosphospecific ACC Ser79 (conc. 1:5000 in 1% BSA) and CPT1B (conc. 1:4000 in 1% BSA). Proteins were visualized by chemiluminiscence (Thermo Scientific, MA, USA) and quantified with a UVP imaging system (UVP, CA, USA). Precision Plus Protein All Blue standards were used as markers of molecular weight (Bio-Rad, CA, USA).
Values derived for quantification of immunoblotting for each protein target were normalized to the total amount of protein loaded for each sample, using the Stain Free Technology approach previously described (16, 17) .