Invitrogen

NEDD4 Polyclonal Antibody

Advanced Verification

This Antibody was verified by Knockdown to ensure that the antibody binds to the antigen stated. View Details

5 Published Figures

3 References

View all (28) NEDD4 antibodies

Datasheet

Protocols

Questions & Answers

Tech Support

Datasheet

Protocols

Questions & Answers

Tech Support

Product Image Gallery

NEDD4 Antibody in Immunocytochemistry (ICC/IF) — Immunofluorescence analysis of NEDD4 was performed using 70% confluent log phase NIH/3T3 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with NEDD4 Rabbit Polyclonal Antibody(Product # PA5-17463) at 1:50 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing Cytoplasmic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

NEDD4 Antibody in Western Blot (WB) — Knockdown of NEDD4 was achieved by transfecting Caco-2 with NEDD4 specific siRNAs (Silencer® select Product # s9416). Western blot analysis (Fig. a) was performed using whole cell extracts from the NEDD4 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with NEDD4 Polyclonal Antibody (Product # PA5-17463, 1:2000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25µg/ml, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to NEDD4.

NEDD4 Antibody — Nedd4 negatively regulates VDAC2/3 stability as the specific E3 ubiquitin ligase. a Nedd4 decreased VDAC2/3 protein in a dose-dependent manner. A375 cells were transfected with Flag-Nedd4 (0, 1.5, and 6?µg) or Flag-Nedd4C867S (6?µg). The protein expression level of VDAC2/3 was assayed by western blot. Nedd4WT can destabilize VDAC2/3, but Nedd4C867S cannot affect the stability of VDAC2/3. b Knockdown of Nedd4 stabilizes VDAC2/3. A375 cells were transfected with control shRNA or Nedd4 shRNAs for 36?h, then treated with DMSO or Erastin (5?µM) for 12?h. The protein levels of VDAC2, VDAC3, and Nedd4 were analyzed by western blot. c Nedd4 ubiquitylates VDAC2/3 in vivo. A375 cells were transfected with indicated DNA constructs for 48?h and treated with MG132 (50?mM) for 4?h before harvest. Cell lysates were immunoprecipitated with anti-Myc and analyzed by immunoblotting with indicated antibodies. d Knockdown of Nedd4 reduced the ubiquitination of VDAC2/3 in vivo. A375 cells were transfected with indicated DNA constructs for 36?h, then treated with DMSO or erastin (5?µM) for 8?h. Before cell harvest, MG132 (50?mM) was added into the medium for 4?h. Cell lysates were immunoprecipitated with anti-Myc and analyzed by immunoblotting with indicated antibodies. e Nedd4 ubiquitylates VDAC2/3 in vitro. Purified VDAC2 and VDAC3 proteins were ubiquitylated in the presence of purified Nedd4 in vitro. See “Methods” for further details. After in vitro ubiquitylation reaction, samples were analyze {KD}

NEDD4 Antibody in Immunohistochemistry (IHC) — Fig. 7 Knockdown of Nedd4 suppresses erastin resistance in vitro and in vivo. a , b Colony formation assay of indicated A375 ( a ) and G-361 ( b ) cells. Cells were treated with erastin (5 uM) for 24 h and were grown without erastin for 10 days. For each cell line, all dishes were fixed, stained, and photographed at the same time. c , d Knockdown of Nedd4 enhanced erastin-induced ferroptosis in vivo. The 7-week-old immunodeficient nude mice (eight mice per group) were injected subcutaneously with indicated A375 cells (5 x 10 6 cells per mouse) and treated with erastin (15 mg per kg intraperitoneal, twice every other day) when the tumor volume reached 50 mm 3 . Tumor volume was calculated every 4 days ( c ), and the tumor mess was measured at day 20 ( c ). The relative levels of MDA and GSH of indicated tumors were measured ( d ). e Immunohistochemistry analysis of VDAC3, Nedd4, and 4HNE of tumor xenografts. Scale bar, 50 um. The experiment was repeated twice independently with similar results. f Schematic depicting the regulation of VDAC2/3 by FOXM1 and Nedd4 during ferroptosis in melanoma cells. Erastin activates FOXM1 to induce the transcription of Nedd4. Increased level of Nedd4 degrades VDAC2/3 and suppresses ferroptotic cell death. Data are mean +- SD from three independent experiments. Comparisons were made using Student's t test. * p < 0.05.

NEDD4 Antibody in Western Blot, Immunoprecipitation (WB, IP) — Fig. 2 Identification of Nedd4 as a VDAC2/3 interacting protein. a The PPxY motifs of VDAC2 (PPSY) and VDAC3 (TPxY) are conserved among different species. See also Fig. S2A , B . b Interaction between endogenous VDAC2, VDAC3, and Nedd4 under basal condition and erastin treatment. A375 cells were treated with DMSO or erastin (5 uM) for 4 h, then MG132 (50 mM) was added into the culture medium for an additional 4 h. Whole-cell lysates (WCL) were used for IP with control serum (IgG) or anti-Nedd4 antibody, followed by immunoblotting (IB) with the indicated antibodies. c Nedd4 directly interacts with VDAC2/3 in vitro. Purified GST (control) or increasing amount of GST-Nedd4 were mixed with VDAC2 or VDAC3 and subjected to GST-pulldown followed by IB for VDAC2 or VDAC3. Efficient VDAC2/VDAC3-Nedd4 interaction was detected in a dose-dependent manner. Coomassie blue stains show the input of each purified proteins used for binding. d The PPxY mutants of VDAC2 and VDAC3 are defective in Nedd4 binding. A375 cells were transfected with indicated DNA constructs for 48 h and treated with MG132 (50 mM) for 4 h. WCL were immunoprecipitated with anti-Flag followed by IB with anti-HA. e Schematic representation of WT Nedd4 and its deletion mutants, and summary of their interactions with VDAC2/3. Plus indicates strong binding; minus indicates no binding. f The WW domain of Nedd4 is essential for interaction with VDAC2/3. HA-VDAC2 or HA-VDAC3 was cotransfected with various Flag-Nedd4 deletion mu

Product Details

Applications

Tested Dilution

Publications

Western Blot (WB)

1:1,000

View 1 publication 1 publication

Immunohistochemistry (IHC)

View 1 publication 1 publication

Immunocytochemistry (ICC/IF)

1:50

Flow Cytometry (Flow)

1:50

Immunoprecipitation (IP)

View 1 publication 1 publication

Product Specifications

Species Reactivity

Human, Mouse, Non-human primate, Rat

Published species

Not Applicable

Host/Isotype

Rabbit / IgG

Class

Polyclonal

Type

Antibody

Immunogen

Synthetic peptide corresponding to human NEDD4 protein

Conjugate

Unconjugated

Form

Liquid

Purification

Antigen affinity chromatography

Storage buffer

0.01M HEPES, pH 7.5, with 0.15M NaCl, 100µg/mL BSA, 50% glycerol

Contains

no preservative

Storage conditions

-20°C

RRID

AB_10976819

Product Specific Information

It is not recommended to aliquot this antibody.

Target Information

The E3 ubiquitin-protein ligase NEDD4 is a Ubl conjugation pathway protein. It contains one HECT (E6AP-type E3 ubiquitin-protein ligase) domain and four WW domains. It is involved in the pathway leading to the degradation of VEGFR-2/KDFR, independently of its ubiquitin-ligase activity. It acts as a potential proto-oncogene that negatively regulates PTEN via ubiquitination, a paradigm analogous to that of Mdm2 and p53. It is also reported to be involved in the budding of many viruses and is the regulator of epithelial Na+ channels, which lead to arterial hypertension. NEDD4 protein is localized to the sarcolemmal region of muscle fibers. Increase in NEDD4 protein expression leads to unloaded and denervated muscles as well as a decrease in Notch1 expression. Hence it acts as down regulator of Notch-1 gene expression.

For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.

Bioinformatics

Protein Aliases: Cell proliferation-inducing gene 53 protein; E3 ubiquitin-protein ligase NEDD4; HECT-type E3 ubiquitin transferase NEDD4; NEDD-4; Neural precursor cell expressed developmentally down-regulated protein 4; neural precursor cell expressed, developmentally down-regulated gene 4; neural precursor cell expressed, developmentally down-regulated gene 4A; neural precursor cell expressed, developmentally down-regulted gene 4; OTTHUMP00000217840; OTTHUMP00000217841; OTTHUMP00000217842; receptor-potentiating factor 1

Gene Aliases: AA959633; AL023035; AU019897; E430025J12Rik; KIAA0093; mKIAA0093; Nedd-4; NEDD4; NEDD4-1; Nedd4a; PIG53; RPF1

UniProt ID: (Human) B4E2R7, (Rat) Q62940, (Mouse) P46935

Entrez Gene ID: (Human) 4734, (Rat) 25489, (Mouse) 17999

Molecular Function: ubiquitin-protein ligase