Sc 19857

Western blots on protein extracts from pulverized tissue were performed as previously described (De Bilbao et al., 2009 (link)). Protein samples were loaded at 30 μg/20 μl, after migration proteins were transferred by semi-dry transfer (De Bilbao et al., 2009 (link)). Membranes were pre-incubated with 1% casein (Vectorlab), then incubated 2 h with primary antibody uncoupling protein-1 (UCP1) dilution 1/5000 (cat. no. UCP11, Alpha Diagnostics), SIRT1 dilution 1/200 (sc-19857, Santa Cruz), FXR 1/200 dilution (sc-13063, Santa Cruz), and beta-actin dilution 1/1000 (Cat No. 4970—Cell Signaling). Secondary antibody LI-COR anti-rabbit (1/15000) or anti-goat (1/15000) were used to detect bands (De Bilbao et al., 2009 (link)). The signals were visualized with the use of Odyssey Infrared Imaging System (LI-COR Biosciences, Bad Homburg, Germany).

Tissue protein extracts underwent gel electrophoresis and proteins were then transferred to membranes (Arsenijevic et al., 2015 (link)). Membranes were pre-incubated with 1% casein (Vectorlab) for 2 h and then rinsed. Membranes were then incubated 2 h with one of the following primary antibodies—uncoupling protein-1 (UCP1) dilution 1/5000 (cat. no. UCP11, Alpha Diagnostics), SIRT1 dilution 1/200 (sc-19857, Santa Cruz), farnesoid x receptor (FXR) dilution 1/200 (sc-13063, Santa Cruz), and beta-actin dilution 1/1000 (Cat No. 4970–Cell Signaling). Secondary antibody LI-COR anti-rabbit (dilution 1/15000) or anti-goat (dilution 1/15000) were used to detect bands (de Bilbao et al., 2009 (link)). The signals were visualized with the use of Odyssey Infrared Imaging System (Li-Cor Biosciences, Bad Homburg, Germany).