Ab70784

After six weeks of drug therapy, all mice were euthanized by intraperitoneal injection with 0.1% pentobarbital sodium. The adipose tissue from the other three groups of mice were removed and stored at −80°C to examine the protein expressions of Insig-1, SCAP, and SREBP-1c. The protocol for western blotting was previously described by us [17 (link)]. Primary antibodies including Insig-1 (Abcam, ab70784, 1 : 200), SREBP-1c (Abcam, ab28481, 1 : 1000), SCAP (Abcam, ab19013, 1 : 1000), IRS-1 (Abcam, ab52167, 1 : 1000), PI3K (Abcam, ab151549, 1 : 1000), Akt (Abcam, ab8805, 1 : 500), mTORC1(Abcam, ab2732, 1 : 200), PAQR3 (Abcam, ab174327, 1 : 250), and β-actin (Beijing Zhongshan Golden Bridge Biotechnology Co., TA-09, 1 : 1000) were used in this study. Protein expression was detected with an enhanced chemiluminescence detection system (Vigorous, Beijing, China).

Immunohistochemistry was performed as previously detailed [5 (link)] using the following primary antibodies: rabbit anti-CHGA (1:400, SP-1, ImmunoStar, Hudson, WI, USA), guinea pig anti-glucagon (1:2500, M8707, Euro-Diagnostica, Malmö, Sweden), goat anti-GIP (1:500, sc-23554, Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-INSIG1 (1:100, ab70784, Abcam, Cambridge, UK) rabbit anti-SCD1 (1:250, #2794, Cell Signalling Technology, Danvers, MA, USA), rabbit anti-SQLE (1:100, Life Technologies, Waltham, MA, USA) and rabbit anti-SREBP2 (1:1000, ab28482, Abcam, Cambridge, UK). The secondary antibodies (dilution 1:400) used were as follows: Cy^TM2 Affinipure donkey anti-rabbit IgG (711225152), Cy^TM2 Affinipure donkey anti-guinea pig IgG (706225148), Cy^TM5 Affinipure donkey anti-guinea pig IgG (706175148) and Cy^TM2 Affinipure donkey anti-goat IgG (705225147; all Jackson ImmunoResearch Europe, Ely, UK). Primary and secondary antibodies were diluted in 0.25% BSA and 0.25% Triton X-100 in PBS. Antibodies were validated by staining alongside a negative control (0.25% BSA and 0.25% Triton X-100 without the antibody) as well as testing in positive control tissue (e. g. liver for SCD).