Anti cd36

Cells were lysed in boiled Sample Buffer 1x (50 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 100 mM DTT). In total, 30 μg of total protein extracts were resolved on 8% SDS-PAGE and electrically transferred onto poly(vinylidene difluoride) membranes (PVDF, Bio-Rad Laboratories, Hercules, CA, USA) membranes. Membranes were blocked with TBS-T (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.02% Tween-20) containing 5% skimmed milk (Bio-Rad), for 1 h at room temperature, and then incubated overnight at 4°C with primary antibodies diluted in TBS-T containing 5% milk. The following antibodies were used: mouse monoclonal anti-ICAM-1 (Santa Cruz Biotechnology, Dallas, TX, USA; working dilution 1:500), rabbit polyclonal anti-CD36 (Santa Cruz Biotechnology, working dilution 1:500), mouse monoclonal anti-α-tubulin (Sigma-Aldrich, working dilution 1:10,000). After extensive washing, immunecomplexes were detected with horse-radish peroxidase conjugated species-specific secondary antibodies (Jackson Laboratory, Bar Harbor, ME, USA) followed by enhanced chemiluminescence reaction (Millipore Corporation, Billerica, MA, USA). Proteins detected by immunoblotting were quantified by densitometry (ChemiDoc imaging system, BioRad) and normalized as a function of α-tubulin with Image-Lab 5.0 software (Bio-Rad).

Murine cerebrovascular endothelial cells underwent Western blotting as previously described by Yin et al.^{9 (link)}. Briefly, the cells were lysed via lysis buffer to isolate the total protein. Equal protein amounts were subjected to SDS-PAGE followed by transfer to polyvinyldifluoride (PVDF) membranes. PVDF membranes were then blocked in a blocking solution (5% non-fat milk in Tris-buffered saline) followed by incubation with the following primary antibodies for two hours at room temperature: anti-PPARγ (1:200; catalog # sc-7273, Santa Cruz Biotechnology), anti-CD36 (1:200; catalog # sc-9154, Santa Cruz Biotechnology), and β-actin (1:500; catalog # sc-47778, Santa Cruz Biotechnology). The membranes were then incubated for one hour at room temperature with the appropriate alkaline phosphatase-conjugated secondary antibodies (Promega). The ProtoBlot AP System (Promega) was applied to assess the color reaction according to the kit’s instructions.

The cells were harvested with lysis buffer containing a protein inhibitor cocktail. Protein was quantified by Bio-Rad assay, and 30 μg of total protein was first subjected to SDS-PAGE and then transferred to nitrocellulose membranes. The membranes were incubated with primary rabbit polyclonal anti-CD36, anti-toll-like receptor 4 (TLR4), anti-phospho-IκBα, or anti-IκBα (Santa Cruz Biotechnology) followed by incubation with a peroxidase-conjugated secondary antibody for 1 hour. Equivalence of protein loading and transfer was confirmed by reblotting the samples with anti-β-actin antibody (Santa Cruz Biotechnology). Immune reactive bands were detected by chemiluminescence and quantified by densitometry. Relative quantities of each protein were normalized by β-actin and expressed as fold increase versus control.