Advanced electrochemiluminescence western blot detection kit

The cells were treated with DS (0, 2.5, 5, and 10 µM) for 24 hours and lysed in PRO-PREP protein extraction solution containing phosphatase and protease inhibitors (Roche Diagnostics GmbH, Mannheim, Germany) for 30 minutes at 4°C. Then, they were centrifuged at 4°C at 13,000 rpm for 30 minutes. The whole protein samples (30 mg) were separated on 8% to 10% SDS PAGE and transferred onto polyvinylidene fluoride membranes polyvinylidene fluoride membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were blocked with 5% BSA (AMRESCO, Cleveland, OH, USA) and in TBS with 0.1% Tween 20 (TBS-T) for one hour at room temperature, and then with primary antibodies diluted (1:200 to 1:1,000) in 5% BSA in TBS-T overnight at 4°C. Next, the membranes were washed four times (3 minutes each) with TBS-T. After washing, the membranes were incubated with anti-rabbit/anti-mouse secondary antibody (1:1,000) for one hour at room temperature and the reactions were detected using an Advanced Electrochemiluminescence Western Blot Detection Kit (Amersham, Uppsala, Sweden). The intensities of the protein bands were measured by ImageJ software.

Cells were treated with UA (0, 5, 10, and 20 μM) for 24 to 48 h and lysed in PRO-PREP Protein Extraction Solution containing protease and phosphatase inhibitors (Roche Diagnostics GmbH, Mannheim, Germany) for 30 min at 4°C. Then, cells were centrifuged at 4°C at 13,000 rpm for 30 min. Whole protein samples (40 μg) were separated using 6% to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (PVDF; Bio-Rad Laboratories Inc., Hercules, CA, USA). The membranes were blocked with 5% borine serum albumin (BSA) (AMRESCO, Cleveland, OH, USA) in Tris-buffered saline (TBS) with 0.1% Tween 20 (TBS-T) for 1 h at room temperature, then incubated with primary antibodies (1:500 to 1:1,000) diluted in 5% BSA in TBS-T overnight at 4°C. Next, membranes were washed four times (3 min each) with TBS-T, and then incubated with HRP-conjugated anti-rabbit or anti-mouse secondary antibodies (1:1,000) for 1 h at room temperature. Protein bands were detected using an Advanced Electrochemiluminescence Western Blot Detection Kit (Amersham, Uppsala, Sweden), and their intensities were measured using ImageJ (National Institutes of Health, Bethesda, MD, USA).