Gtx100642

Macrophages from each group were collected and lysed using RIPA lysis buffer, followed by protein quantification utilizing the bicinchoninic acid method. 10 μg total protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for separation and moved to the polyvinylidene difluoride (PVDF) membrane. 5% defatted milk powder in 0.5% TBST was prepared to block the membranes for 2 h. Primary antibodies against p-p65 (ab194926, 1: 1000, Abcam), p65 (ab16502, 1:2000, Abcam), SIRT1 (ab189494, 1:1000, Abcam), LOX-1 (ab214427, 1: 1000, Abcam), CD36 (GTX100642, 1:500, GeneTex, Shanghai) and GAPDH (GTX 100118, 1:10000; GeneTex) were employed to incubate the membrane at 4°C overnight. The membrane was rinsed 3 times by TBST and cultivated with Goat anti-rabbit secondary antibody (ab205718, 1:5000, Abcam) at room temperature for 2 h. The visualization and analysis of protein blots were carried out employing an ECL (Amersham) and Image J (version 1.52; National Institute of Health) [15 (link)].

The mice were euthanized using carbon dioxide overdose. The liver, intestine, and epididymal white adipose tissue (EWAT) were harvested, fixed in 4% formaldehyde, paraffin-embedded, sectioned, and stained using hematoxylin and eosin (H&E) or immunohistochemistry (IHC). IHC was performed as previously described [19 (link)]. The appropriate volume of primary anti-cluster of differentiation 36 (CD36) (1:100, GTX100642, GeneTex) antibody was added to cover the specimen and the samples were incubated at 4 °C overnight. Nuclei were stained with hematoxylin. The images were captured using Pannoramic MIDI II (3DHISTECH Ltd., Budapest, Hungary).