Article Title: Sodium Citrate Increases Expression and Flux of Mg2+ Transport Carriers Mediated by Activation of MEK/ERK/c-Fos Pathway in Renal Tubular Epithelial Cells
Figure Lengend Snippet: Activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and epidermal growth factor receptor (EGFR) by SC. ( A ) NRK-52E cells were incubated with vehicle (veh), 10 μM SC, 10 μM diphenyleneiodonium (DPI), or 10 μM 5-( N -ethyl- N -isopropyl) amiloride (EIPA) for 24 h. Quantitative real-time PCR was performed using primer pairs of TRPM6 and β-actin. ( B ) Cells were incubated with vehicle (vih), 10 μM SC, 10 μM DPI, or 10 μM EIPA for 15 min. Cytoplasmic extracts were immunoblotted with anti-p-ERK1/2 or anti-ERK1/2 antibody. ( C ) Cells were incubated with vehicle (veh), 10 μM SC, 10 μM DPI, or pH 7.6 for 15 min. ROS production was assessed by 2′,7′-dichlorofluorescin diacetate (DCFDA) fluorescence measurement and represented as a percentage of vehicle. ( D ) Cells were incubated with vehicle (veh), 10 μM SC or pH 7.6 for 15 min. Cytoplasmic extracts were immunoprecipitated with anti-NADPH oxidase (NOX) 4 antibody. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the membrane was blotted with anti-p22phox or anti-NOX4 antibody. ( E , F ) Cells were incubated with vehicle (veh), 10 μM SC, 10 μM DPI, 10 μM EIPA, or 10 μM erlotinib (ERL) for 15 min. Cytoplasmic extracts were immunoblotted with anti-p-EGFR, anti-EGFR, anti-p-ERK1/2, or anti-ERK1/2 antibody. The expression levels were represented relative to the values in vehicle. n = 3–4. ** p
Article Snippet: Anti-actin, anti-phosphorylated ERK1/2 (p-ERK1/2), anti-c-Fos, and anti-p22phox antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Techniques: Activation Assay, Incubation, Real-time Polymerase Chain Reaction, Fluorescence, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, SDS Page, Expressing