Quantity one image analyzer software program

Livers (0.1 g) were homogenized in 0.1 mg/ml phenylmethylsulfonyl fluoride-containing ice-cold RIPA buffer (1 ml) and then the lysates were clarified by centrifugation (12,000 g, 4°C, 15 min), after that the supernatant was collected and stored at −80°C. The samples were denatured with loading buffer (99°C, 10 min), then the equal protein were separated by SDS-PAGE gel electrophoresis and transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was probed with primary antibody according to the dilution ratio provided by manufacturers overnight at 4°C, and then incubated with secondary antibody according to the dilution ratio provided by manufacturers at room temperature for 60 min. The immunoreactivity was detected using the ChemiDoc XRS + detection system (ECL, Bio-Rad, United States). The densitometric analysis was performed with Quantity One^® Image Analyzer software program (Bio-Rad). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalization.

The concentrations of total protein extracted with the RIPA reagent were detected by the BCA protein assay kit. Protein extracts were diluted, boiled with SDS PAGE loading buffer at 95 °C for 10 min and then were subjected to protein separation by loading onto 10% SDS PAGE. Proteins in the gel were transferred to a PVDF membrane. The membranes were blocked with 5% skimmed milk powder in tris buffered saline with 0.05% Tween 20 (TBS-T) for 2 h at room temperature. After a short wash with TBS-T, the membranes were incubated with specific primary antibodies which were diluted in TBS-T by 200 to 5,000 folds overnight at 4 °C. The membranes were washed four times with TBS-T and then were incubated with secondary antibody diluted in TBS-T by 2500- or 5000-fold for 1 h at room temperature. After washing four times with TBST, the detection of proteins was performed using the ChemiDoc XRS + detection system (ECL, Bio-Rad). The immunoblots were analyzed with the Quantity One^® Image Analyzer software program (Bio-Rad). β-actin was used as an internal reference for examined proteins except membrane UT-A1. Equal loading of protein amounts while evaluating membrane UT-A1 was demonstrated using Ponceau staining.

Cells cultured in 10-cm dishes were washed thrice with PBS and then lysed in Radio-Immune Precipitation Assay (RIPA) lysis buffer containing 1% PMSF on ice for 30 min. Following centrifugation (15,000× g, 10 min, 4 °C), the supernatants were incubated at 95 °C for 10 min in 5×SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) loading buffer. After protein separation through electrophoresis on a 15% SDS-PAGE gel, proteins in the gel were transferred to a Polyvinylidene difluoride (PVDF) membrane. The membrane was blocked using 5% nonfat dried milk in TBS-T (10 mM Tris-HCl, pH7.8, 150 mM NaCl and 0.05% Tween-20 (v/v)) for at least 2 h at room temperature. After a short wash with the TBS-T, the membrane was incubated with a primary antibody diluted in the TBS-T by 1000- to 5000-fold for at least 3 h at room temperature. The membrane was washed thrice with the TBS-T and then was incubated with a secondary antibody diluted in the TBS-T by 2500- to 5000-fold for 1 h at room temperature. Following three washings with the TBS-T, protein signals were detected using ChemiDoc XRS⁺ detection system (ECL, Bio-Rad, Hercules, CA, USA). The Quantity One^® Image Analyzer software program (Bio-Rad) was used for quantitative densitometric analysis.