Glyceraldehyde 3 phosphate dehydrogenase antibody

Lipofectamine™ 2000 transfection reagent (Invitrogen) was used to transfect miRNA and construct the cells. AC16 cells were transfected with 3.0 μg pcDNA3.1‐RAGE‐rs2070600‐G or ‐A plasmid and 100 nmol of chemically synthesized has‐miR‐125a‐3p (RIBOBIO Co., Ltd, Guangzhou, China), miRNA inhibitor(s), or miR‐negative control (RIBOBIO Co., Ltd, Guangzhou, China) (Supporting Information, TableS5). After 48 h, the cells were washed and homogenized with lysis solution (50 mM Tris‐Cl, pH 8.0; 150 mM NaCl; 0.02% sodium azide; 0.1% SDS; 1 μg/mL aprotinin; 1% Nonidet P‐40; and 0.5% sodium deoxycholate) containing protease inhibitors (100 μg/mL phenylmethylsulfonyl fluoride, 2 μg/mL aprotinin, and 2 μg/mL leupeptin). Samples were then centrifuged at 12 000 × g for 20 min at 4°C, and supernatants were collected. Cell lysates were resolved by 10% SDS‐polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. After blocking with 5% nonfat milk, blots were probed with RAGE antibody (ABclonal, China, A13264), anti‐FLAG antibody (Cell Signalling Technology, USA, 147935), and glyceraldehyde 3‐phosphate dehydrogenase antibody (Santa Cruz Biotechnology, USA, sc‐293,335). Bands were visualized using enhanced chemiluminescence reagents (Pierce Chemical, Rockford, IL) and quantified by densitometry.

GRK2 levels were evaluated at admission and after 8 weeks of treatment for all patients as described previously.29 (link) The peripheral blood mononuclear cells were isolated from the whole blood samples by density-gradient centrifugation on Ficoll-Hypaque (Sigma-Aldrich Co., St Louis, MO, USA),30 (link) and stored at −80°C until the day of assay. Immunodetection of GRK2 levels was performed by Western blot using polyclonal anti-GRK2 antibodies31 (link) (Santa Cruz Biotechnology, Dallas, TX, USA), glyceraldehyde 3-phosphate dehydrogenase antibody (1:2,000; Santa Cruz Biotechnology) was used as an internal loading control. The membranes were incubated with antibody overnight, and then incubated with the corresponding secondary antibody for 1.5 hours. The results were analyzed using Quantity One ImageJ software (National Institute for Health, Bethesda, MD, USA) to quantify the levels of the GRK2 protein.