NADPH oxidase 4/NOX4 Recombinant Rabbit Monoclonal Antibody [SY0214]

Western blot analysis of NADPH oxidase 4/NOX4 on different lysates with Rabbit anti-NADPH oxidase 4/NOX4 antibody (ET1607-4) at 1/2,000 dilution.

Lane 1: SH-SY5Y cell lysate
Lane 2: U-2 OS cell lysate

Lysates/proteins at 15 µg/Lane.

Predicted band size: 67 kDa
Observed band size: 67 kDa

Exposure time: 3 minutes;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1607-4) at 1/2,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:50,000 dilution was used for 1 hour at room temperature.

☑ Knockdown (KD)

Western blot analysis of NOX4 with anti-NOX4 antibody [SY0214] (ET1607-4) at 1/1,000 dilution.

Lane 1: Wild-type SH-SY5Y whole cell lysate (20 µg).
Lane 2/3: NOX4 fragment 1 knockdown SH-SY5Y whole cell lysate (20 µg).
Lane 4/5: NOX4 fragment 2 knockdown SH-SY5Y whole cell lysate (20 µg).

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM in TBST for 1 hour at room temperature. The primary antibody (ET1607-4, 1/1,000) and Loading control antibody(Rabbit anti-HSP90, ET1605-56, 1/10,000) were used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG-HRP Secondary Antibody (HA1001) at 1/200,000 dilution was used for 1 hour at room temperature.

NADPH oxidase 4/NOX4 was immunoprecipitated in 0.2mg SH-SY5Y cell lysate with ET1607-4 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using ET1607-4 at 1/1,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: SH-SY5Y cell lysate (input)
Lane 2: Rabbit IgG instead of ET1607-4 in SH-SY5Y cell lysate
Lane 3: ET1607-4 IP in SH-SY5Y cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 24 seconds; ECL: K1801

Immunocytochemistry analysis of SH-SY5Y cells labeling NADPH oxidase 4/NOX4 with Rabbit anti-NADPH oxidase 4/NOX4 antibody (ET1607-4) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-NADPH oxidase 4/NOX4 antibody (ET1607-4) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

Flow cytometric analysis of SH-SY5Y cells labeling NADPH oxidase 4/NOX4.

Cells were fixed and permeabilized. Then stained with the primary antibody (ET1607-4, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

Immunohistochemical analysis of paraffin-embedded human kidney tissue using anti-NADPH oxidase 4/NOX4 antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes.The tissues were blocked in 1% BSA for 30 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1607-4, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Immunohistochemical analysis of paraffin-embedded rat kidney tissue using anti-NADPH oxidase 4/NOX4 antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes.The tissues were blocked in 1% BSA for 30 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1607-4, 1/200) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.