Figure 2. Western blot analysis of NADPH oxidase 4 using NADPH oxidase 4. Electrophoresis was performed on a 5-20% SDS- PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50ug of sample under reducing conditions. Lane 1: rat kidney tissue lysates, Lane 2: rat NRK whole cell lysates, Lane 3: mouse kidney tissue lysates, Lane 4: mouse HEPA1-6 whole cell lysates, Lane 5: mouse SP20 whole cell lysates. After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with rabbit NADPH oxidase 4 at 0.5ug/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat rabbit IgG-HRP secondary antibody at a dilution of 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit with Tanon 5200 system. A specific band was detected for NADPH oxidase 4 at approximately 85KD. The expected band size for NADPH oxidase 4 is at 67kD.

Figure 3. Immunohistochemistry analysis of NADPH oxidase 4 using NADPH oxidase 4. NADPH oxidase 4 was detected in paraffin- embedded section of human renal cancer tissue. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1ug/ml NADPH oxidase 4 Antibody overnight at 4°C. Biotinylated goat rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) with DAB as the chromogen.