Af2519

Freshly isolated mouse TBC were lysed using a micro-potter in 20 µL of TSE buffer (50 mM Tris HCl, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P40, 5 µL/mL protease inhibitors (Sigma)) [25 (link)]. Samples were stored on ice for 30 min, and then centrifuged (10,000 g, 10 min, 4°C). Lysates were used immediately or stored at −80 °C until the assay. Protein concentrations in homogenates were assayed using the BCA assay (Sigma, Saint Quentin-Fallavier, France). Denatured proteins (25 µg) were separated by SDS-PAGE (8%) and transferred to a polyvinylidene difluoride membrane. After being blocked for 3 h using a TBS buffer containing 5% BSA and 0.05% Tween-20, the membrane was incubated overnight with either of the antibodies: anti-CD36 antibody (R&D Systems, AF2519; 1:1000), anti-GPR120 antibody (Abcam, Paris, France, ab97272; 1:500), anti-α-gustducin antibody (Santa Cruz, Heidelberg, Germany, sc-395; 1:200) and anti-β-actin antibody (Santa Cruz, Heidelberg, Germany, sc-47778; 1:5000). The α-gustducin was used as an internal reference protein. After a set of washes, the appropriate peroxidase-conjugated secondary antibody was added. Antibody labeling was detected by chemiluminescence (Clarity, Bio-Rad, Marnes-la-Coquette, France).

Immunoblot analysis was performed as described previously^{66 (link)} using antibodies against p-p65 NFκB and total p65 NFκB (Cell Signaling Technology 3033, 8242), CD36 (R&D Systems AF2519) and GLUT1 (Abcam 115730). Protein levels were normalized against β-actin (Abcam 8226).