Hsl antibody

Western blot was conducted to assess the protein of interest [23 (link)]. Snap-frozen samples were homogenized in a modified radioimmunoprecipitation assay buffer and followed by centrifugation to extract supernatants. An equal amount of protein was loaded on SDS-PAGE, which was transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The proteins were then immunoblotted with various primary and secondary antibodies, and visualized on a Li-COR Imager System (Li-COR Biosciences, Lincoln, NE, USA). The protein antibodies are listed as follows: UCP1 antibody (1:500, abcam, ab23841), Mitochondrial total OXPHOS protein antibody set (Abcam, ab110413), phospho-HSL antibody (pHSL) (1:1000, Cell Signaling 4126), HSL antibody (1:1000, Cell Signaling 4107), α-Tubulin antibody (1:1000, Advanced BioChemicals, ABCENT4777, Lawrenceville, GA, USA) and the secondary antibody Alexa Fluor 680 (ThermoFisher Scientific).

Immunoblotting was performed as previously described [32 (link),61 (link)] with rabbit polyclonal Phospho-Akt (Ser473) antibody (1:1000, #9271; Cell Signaling Technology, Tokyo, Japan), Akt antibody (1:1000, #9272; Cell Signaling Technology, Tokyo, Japan), ATGL antibody (1:1000, #2138; Cell Signaling Technology, Tokyo, Japan), Phospho-HSL (S563) antibody (1:1000, #4139; Cell Signaling Technology, Tokyo, Japan), HSL antibody (1:1000, #4107; Cell Signaling Technology, Tokyo, Japan), Plin5 antibody (1:1000, GP-31; PROGEN Biotechnik, Germany), and GAPDH antibody (1:5000, #2118; Cell Signaling Technology, Tokyo, Japan). The signals were detected using chemiluminescence.