Tirap

Specific siRNA targeting TRIF (sc-154266), TRAM (sc-44748), TIRAP (sc-44740), Myd88 (sc-35987), lipoprotein lipase (LPL) (sc-44900), glycosylphosphatidylinositol high density binding protein 1 (GPI-HBP1) (sc-145686), clathrin (sc-35067), and caveolin-1 (sc-29241) were purchased from Santa Cruz Biotechnology. Scramble siRNA (sc-37007) used as control. Cells were transfected with the indicated siRNA duplex targeting constructs (50 nM) and HiPerFect Transfection Reagent (QIAGEN, Hilden, Germany). After incubation for 24 h, downregulation of target gene expression was evaluated by RT-PCR.

After treatment, SH-SY5Y cells were solubilized in lysis buffer containing 20 mM Tris–HCL (pH 7.5), 137 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 1% nonidet P40, 10% glycerol, protease inhibitors (0.35 mg/ml PMSF, 2 µg/ml leupeptin and 2 µg/ml aprotinin) and phosphatase inhibitors (10 mM sodium fluoride, 1 mM sodium orthovanadate, 20 mM sodium β-glycerophosphate and 10 mM benzamidine). Lysates protein concentration was determined using the BCA Protein Assay Kit (Thermo Scientific, Illkirch, France) and analyzed by Western blot. Briefly, proteins were subjected to SDS-PAGE and transferred onto Immobilon-FL membrane (Life Technologies, Illkirch, France). Blots were then blocked with 5% Bovine Serum Albumin fraction V (BSA) (Euromedex, souffelweyersheim, France) and incubated with primary antibodies raised against p-AKT, AKT, p-P38MAPK, P38MAPK, pERK1/2, ERK1/2, Insulin receptor (IR) β subunit, β-tubulin (Cell signaling, Danvers, MA, USA), Toll-like receptor 4 (TLR4), MyD88, TIRAP (Santa Cruz Biotechnology, Dallas, TX, USA) and resistin (Abcam, Cambridge, UK) overnight at 4 °C. The immunoblots were washed with PBS, incubated with the appropriate secondary antibody labeled with Alexa 488 (Life Technologies, Illkirch, France) then scanned and quantified using the Brucker Molecular Imaging System 4000MM Pro (Billerica, MA, USA).