Oxidative Stress and Adipocyte Biology: Focus on the Role of AGEs

Figure 4

Glycated albumin induces CD36 expression in SW872 adipocytes. (a) SW872 adipocytes were incubated for 24 hr in the absence (PBS) or the presence of 50 μM native human serum albumin (HSA) or AGEs constituted by methylglyoxal- (MGO) modified HSA. The relative quantification (% of fluorescent cells versus control PBS) of CD36 receptors was determined by employing a Becton Dickinson FACScan (BD Biosciences, San Jose, CA) after staining with a primary human CD36 antibody (1 : 50) for 1 hr, followed by incubation with the secondary Alexa Fluor 488 conjugated anti-rabbit (1 : 100) and PE-conjugated anti-mouse antibodies (1 : 100) for an additional hour. Cells were washed with a 1% PBS/BSA blocking buffer between each incubation step. (b) For the CD36 Western blot, 20 μg proteins were isolated from SW872 cell lysates (with different treatments as indicated), separated by SDS-PAGE, and transferred onto a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA) using a liquid transfer system. Membranes were soaked overnight with blocking buffer (PBS/0.1% Tween/1% BSA) and subsequently incubated in blocking buffer with a primary human CD36 antibody (1 : 200) for 2 hr, followed by incubation with a secondary HRP-conjugated sheep anti-mouse IgG antibody (1 : 2,000). Membranes were washed with a blocking buffer (0.1% PBS/1% Tween) between different incubation steps. Protein bands were detected by standard ECL methods (Amersham Biosciences, Amersham, UK) and visualized with a Kodak 2000R Image station (Eastman Kodak, Rochester, NY), and routine densitometric analysis was performed for quantification.

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