Effect of High glucose on HuR protein expression in RAW 264.7 cells

Cell culture and high glucose treatment: RAW 264.7 cells (mouse monocyte/macrophage cell line, ATCC TIB-71) were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Life technologies, Grand Island, NY) with 10% Fetal Bovine Serum (FBS, ATCC, Manassas, VA) and 1% Penicillin-Streptomycin (Life Technologies) and were incubated in a humidified chamber at 37°C with 5% CO₂. Cells were cultured in either high glucose (HG, 30mM) or normal glucose (NG, 5mM) media.Protein extraction and western blot analysis: Protein lysates were prepared using Cell Lysis Buffer 1 (Part No.: 890713, R & D Systems). Equal amounts of proteins from cells were separated by 10% Mini-PROTEAN TGX Stain-Free Protein Gels (Cat# 4568034, Bio-Rad) and blotted on to Trans-Blot Turbo Mini polyvinylidenedifluoride (PVDF) membranes (Cat# 1704156, Bio-Rad). The blots were incubated with antibodies against HuR (1:100, Cat# sc-5261, Santa Cruz Biotechnology Inc.) and GAPDH (1:100, Cat# MA5-15738, Thermo Fisher Scientific) and developed with an enhanced chemiluminescence detection system (Bio-Rad, ChemiDoc Touch Imaging System). The densitometry of the bands from the western blot was analyzed using NIH-ImageJ software.Our observation: RAW 264.7 cells treated with HG (30mM) showed an increase in HuR protein levels as compared to cells treated with NG (5mM).