Ecl immunoblotting detection reagent

Cells were lysed with SDS sample buffer. Equal amounts of cell lysates were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA), which was then incubated with the respective primary antibodies, followed by incubation with peroxidase-conjugated secondary antibody. The primary antibodies used were mouse monoclonal antibodies against NS3, NS5A and GAPDH (Chemicon International, Temecula, CA, USA). The respective proteins were visualized using ECL immunoblotting detection reagents (GE Healthcare).

Western blots were performed to assess the renal cortex and medulla protein levels of eNOS and iNOS. Short isoform-specific primary antibodies to eNOS (1 : 1000 dilution, SC-654, Santa Cruz Biotech, Santa Cruz, CA) and iNOS (iNOS-A, 1 : 2000, Alpha Diagnostics International, San Antonio, TX) were used. Goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1 : 2000 dilution, number 20320, Alpha Diagnostics International, San Antonio, TX) was subsequently applied. β-Actin was measured as an internal control using a monoclonal primary antibody (1 : 2000 dilution, A2228, Sigma-Aldrich Corporation, St. Louis, MO) and horseradish peroxidase-conjugated goat anti-mouse secondary antibody (1 : 30 000 dilution, A9044, Sigma-Aldrich Corporation, St. Louis, MO). Membranes were detected with ECL immunoblotting detection reagents (GE Healthcare, Piscataway, NJ) and bands were quantified using a Chemi Doc chemiluminiscent detection system and Quantity One software (Bio-Rad, Richmond CA). The results were expressed as NOS/β-actin density ratio.

Immunoblotting was performed as described earlier (Qi et al., 2009 (link)). Frozen tissue samples were sonicated in 1% SDS, transferred to Eppendorf tubes and boiled for additional 10 min. Small aliquots of the homogenate were retained for protein determination using the bicinchoninic acid protein assay method (Pierce, Rockford, IL, United States). Equal amounts of protein (20 μg) were loaded onto 12% acrylamide gels, and the proteins were separated by SDS-PAGE and transferred to Immobilon^®-P Polyvinylidene Difluoride membranes (Sigma). Immunoblotting was performed on the membranes using P-Ser¹⁹-TH (Merck Millipore, Billerica, MA, United States), P-Ser³¹-TH (Millipore), P-Ser⁴⁰-TH (Millipore), P-Ser⁸⁴⁵-GluA1 (UBI), and antibodies, which are not phosphorylation state-specific to estimate total levels of TH (Millipore) and GluA1 (UBI). The antibody binding was detected by incubation with goat anti-mouse or anti-rabbit horseradish peroxidase-linked IgG (1:6000–8000 dilution) and detected using ECL immunoblotting detection reagents (GE Healthcare, Little Chalfont, United Kingdom).