Total pka

Proteins were isolated from tissues with lysis buffer [100 mM tris-HCl (pH 6.8), 2.0% SDS, 20% glycerol, 0.02% bromophenol blue, 5% 2-mercaptoethanol, 100 mM NaF, and 1 mM Na₃VO₄]. The concentration of homogenized protein lysates was determined by the Bradford method (Bio-Rad). The following antibodies were used for the detection of target proteins: UCP1 (#14670), phospho-PKA (#5661), total PKA (#4782), phospho-AMPK (#2535), and total AMPK (#2793) were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against glucose transporter 4 (GLUT4; sc-1607), apelin (sc-293441), and HIF-1α (sc-13515) were purchased from Santa Cruz Biotechnology (Dallas, TX). PRDM16 (PA5-20872; Thermo Fisher Scientific, Rockford, IL) and PGC-1α (no. 66369-1-Ig; Proteintech, Rosemont, IL) were also purchased. Antibodies against β-actin and β-tubulin were obtained from the Developmental Studies Hybridoma Bank (Iowa City, IA). IRDye 680 goat anti-mouse secondary antibody (1:10,000), IRDye 800CW donkey anti-rabbit secondary antibody (1:10,000), and IRDye 800CW donkey anti-goat secondary antibody (1:10,000) were purchased from LI-COR Biosciences (Lincoln, NE). The target proteins were detected using the infrared imaging system (Odyssey, LI-COR Biosciences), and intensity of band was quantified using Image Studio Lite (LI-COR Biosciences) (16 (link)).

The proteins were extracted from cells with different treatments. The proteins were performed with SDS‐PAGE gels and transferred by the PVDF membranes (Millipore, Burlington, MA, USA). After blocking, the membranes were visualized by the ECL system (GE, Chalfont St Giles, England). The EP1–4 antibodies were purchased from Abcam (Cambridge, MA, USA), and the active β‐catenin, total β‐catenin, TGF‐β, p‐PKA, total PKA, p‐AKT, and total AKT antibodies were acquired from Cell Signaling Technology (Beverly, MA, USA). The collagen I and collagen III antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).