Antibody data
- Antibody Data
- Antigen structure
- References [1]
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- Validations
- Western blot [3]
- Immunocytochemistry [2]
- Other assay [5]
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- Product number
- PA5-17463 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- NEDD4 Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Synthetic peptide
- Description
- It is not recommended to aliquot this antibody.
- Reactivity
- Human, Mouse, Rat
- Host
- Rabbit
- Isotype
- IgG
- Vial size
- 100 µL
- Concentration
- 12 µg/mL
- Storage
- -20°C
Submitted references Nedd4 ubiquitylates VDAC2/3 to suppress erastin-induced ferroptosis in melanoma.
Yang Y, Luo M, Zhang K, Zhang J, Gao T, Connell DO, Yao F, Mu C, Cai B, Shang Y, Chen W
Nature communications 2020 Jan 23;11(1):433
Nature communications 2020 Jan 23;11(1):433
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Supportive validation
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- Invitrogen Antibodies (provider)
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- Experimental details
- Western blot analysis of NEDD4 in lysates from NIH/3T3 and C6 cells using NEDD4 polyclonal antibody (Product # PA5-17463).
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- Invitrogen Antibodies (provider)
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- Experimental details
- 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.
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- Experimental details
- Western blot analysis was performed on whole cell extracts (30 µg lysate) of NIH/3T3 (Lane 1), A549 (Lane 2), K-562 (Lane 3), PANC-1 (Lane 4), tissue extracts (30 µg lysate) Mouse Skeletal Muscle (Lane 5) and Rat Skeletal Muscle (Lane 6). The blot was probed with Anti-NEDD4 Polyclonal Antibody (Product # PA5-17463, 1:2000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/ml, 1:4000 dilution). A 110 kDa band corresponding to NEDD4 was observed across the cell lines and tissue extracts tested.
Supportive validation
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- Invitrogen Antibodies (provider)
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- Experimental details
- Immunofluorescent analysis of NEDD4 in untreated C6 cells using a NEDD4 polyclonal antibody (Product # PA5-17463) (green). DNA is labeled using a fluorescent blue dye.
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- Invitrogen Antibodies (provider)
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- Experimental details
- 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.
Supportive validation
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- Invitrogen Antibodies (provider)
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- Experimental details
- 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
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- Fig. 3 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 ug) or Flag-Nedd4 C867S (6 ug). The protein expression level of VDAC2/3 was assayed by western blot. Nedd4 WT can destabilize VDAC2/3, but Nedd4 C867S 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 uM) 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 uM) 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
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- Fig. 4 Nedd4 regulates erastin-induced ferroptosis. a - f Knockdown of Nedd4 enhanced erastin-induced ferroptotic cell death. Indicated cells were treated with erastin (1-20 uM) for 24 h, and cell viability was assayed using a CCK8 kit ( a ). Indicated cells were treated with erastin (5 uM) for 24 h, the lipid ROS level was assessed by flow cytometry using C11-BODIPY ( b ), and the lipid formation was measured by MDA assay ( c ). The accumulation of Fe 2+ was measured by iron detection assay ( d ), concentrations of GSH, and GSSG were detected by relative assay kits ( e ). The expression of Nedd4 was assessed by immunoblotting and compared to actin levels ( f ). Number 1 and black color represent control sh group; number 2 and red color represent Nedd4 sh + Vector group; number 3 and green color represent Nedd4 sh + Nedd4 WT group; number 4 and blue color represent Nedd4 sh + Nedd4 C867S group. g - l Overexpression of Nedd4 suppressed erastin-induced ferroptotic cell death. Indicated cells were treated with erastin (1-20 uM) for 24 h, and cell viability was assayed using a CCK8 kit ( g ). Indicated cells were treated with erastin (5 uM) for 24 h, the lipid ROS level was assessed by flow cytometry using C11-BODIPY ( h ), and the lipid formation was measured by MDA assay ( i ). The accumulation of Fe 2+ was measured by iron detection assay ( j ), concentrations of GSH and GSSG were detected by relative assay kits ( k ). The expression of Nedd4 was assessed by immunoblotting and
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- Fig. 6 Upregulation of Nedd4 by erastin was mediated by FOXM1. a , b Erastin induced the expression of FOXM1 and Nedd4. A375 cells were treated with erastin at different time points (0-12 h) or different concentrations (0-10 uM). Cell lysates were analyzed by immunoblotting with indicated antibodies. The protein levels of FOXM1 and Nedd4 were quantitated by Image J. c Structure of Nedd4 promoter showing the location of two FOXM1 binding sites (in red). d ChIP analysis shows occupancy by FOXM1 on Nedd4 promoter, as indicated, in A375 cells treated with erastin (5 uM) for 8 h. GAPDH serves as a negative control. e Dual-luciferase reporter assay showing the activity of Nedd4 promoter in response to erastin in A375 cells. The Nedd4 promoter reporters were transfected into A375 cells for 24 h, treated by erastin (5 uM) for an additional 12 h, then luciferase activity was measured. f Knockdown of FOXM1 suppressed erastin-induced Nedd4 expression and blocked VDAC2/3 degradation. A375 cells expressing control shRNA or FOXM1 shRNA constructs were treated with DMSO or erastin (5 uM) for 12 h, the protein levels of VDAC2, VDAC3, and Nedd4 were assayed by western blot. g Overexpression of Nedd4 but not the C867S mutant rescues the protein degradation of VDAC2/3 inhibited by FOMX1 knockdown. Cells expressing indicated DNA constructs were treated with DMSO or erastin (5 uM) for 12 h, the protein level of VDAC2/3 was assayed by western blot. h - j Overexpression of Nedd4 or knockdown of VDA
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- 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.