Figure 1. Western blot analysis of NADPH oxidase 4/NOX4 using anti-NADPH oxidase 4/NOX4 antibody
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: human HepG2 whole cell lysates,
Lane 2: human SW620 whole cell lysates,
Lane 3: human HK-2 whole cell lysates,
Lane 4: human HL-60 whole cell lysates,
Lane 5: human 293T whole cell lysates,
Lane 6: human SW579 whole cell lysates,
Lane 7: human SK-OV-3 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 anti-NADPH oxidase 4/NOX4 antigen affinity purified polyclonal antibody at 0.5 ug/mL overnight at 4 degree C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-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/NOX4 at approximately 67KD. The expected band size for NADPH OXIDASE 4/NOX4 is at 67KD.
Figure 2. IHC analysis of NADPH OXIDASE 4/NOX4 using anti-NADPH OXIDASE 4/NOX4 antibody (PA1929).
NADPH OXIDASE 4/NOX4 was detected in paraffin-embedded section of rat kidney tissues. 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 rabbit anti-NADPH OXIDASE 4/NOX4 Antibody overnight at 4 degree C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37 degree C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) with DAB as the chromogen.
Figure 3. Flow Cytometry analysis of U20S cells using anti-NADPH oxidase 4/NOX4 antibody
Overlay histogram showing U20S cells stained (Blue line).The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti- NADPH oxidase 4/NOX4 Antibody (1ug/1x106 cells) for 30 min at 20 degree C. DyLight 488 conjugated goat anti-rabbit IgG (BA1127, 5-10ug/1x106 cells) was used as secondary antibody for 30 minutes at 20 degree C. Isotype control antibody (Green line) was rabbit IgG (1ug/1x106) used under the same conditions. Unlabelled sample (Red line) was also used as a control.
Figure 4. Western blot analysis of NADPH oxidase 4/NOX4 using anti-NADPH oxidase 4/NOX4 antibody
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 lysate.
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 anti-NADPH oxidase 4/NOX4 antigen affinity purified polyclonal antibody at 0.5 ug/mL overnight at 4 degree C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-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. Specific bands were detected for NADPH oxidase 4/NOX4 at approximately 67-80KD. The expected band size for NADPH oxidase 4/NOX4 are at 67-80KD.
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