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).

细胞培养与高糖处理：将RAW 264.7细胞（小鼠单核/巨噬细胞系，美国典型培养物保藏中心（ATCC）TIB-71）培养于达尔伯克改良伊格尔培养基（Dulbecco's Modified Eagle's Medium, DMEM，Life Technologies公司，美国纽约州格兰艾兰）中，添加10%胎牛血清（Fetal Bovine Serum, FBS，美国典型培养物保藏中心（ATCC），弗吉尼亚州马纳萨斯）与1%青霉素-链霉素（Life Technologies公司），随后置于37℃、含5%CO₂的饱和湿度培养箱中孵育。细胞分别培养于高糖（HG，30mM）培养基或正常糖（NG，5mM）培养基中。
蛋白提取与蛋白质印迹（Western Blot）分析：采用细胞裂解液1（货号：890713，R&D Systems公司）制备蛋白裂解液。取等量细胞总蛋白，经10% Mini-PROTEAN TGX免染蛋白凝胶（货号：4568034，Bio-Rad公司）电泳分离，随后转印至Trans-Blot Turbo微型聚偏二氟乙烯（polyvinylidenedifluoride, PVDF）膜（货号：1704156，Bio-Rad公司）。将膜与抗HuR抗体（稀释比例1:100，货号：sc-5261，圣克鲁斯生物技术公司（Santa Cruz Biotechnology Inc.））及抗GAPDH抗体（稀释比例1:100，货号：MA5-15738，赛默飞世尔科技（Thermo Fisher Scientific））孵育，通过增强化学发光检测系统（Bio-Rad ChemiDoc Touch成像系统）完成显影。采用NIH ImageJ软件对蛋白质印迹的条带光密度进行定量分析。
实验观察：与正常糖（5mM）处理的RAW 264.7细胞相比，经高糖（30mM）处理的细胞中HuR蛋白水平显著升高。