- PA5-30382 - Invitrogen Antibodies IFITM3 antibody

Submitted references Monoclonal antibody targeting BDCA2 ameliorates skin lesions in systemic lupus erythematosus.

Furie R, Werth VP, Merola JF, Stevenson L, Reynolds TL, Naik H, Wang W, Christmann R, Gardet A, Pellerin A, Hamann S, Auluck P, Barbey C, Gulati P, Rabah D, Franchimont N
The Journal of clinical investigation 2019 Mar 1;129(3):1359-1371

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot using IFITM3 Polyclonal Antibody (Product # PA5-30382). Sample (30 µg of whole cell lysate). Lane A: HeLa . 15% SDS PAGE. IFITM3 Polyclonal Antibody (Product # PA5-30382) diluted at 1:1,000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: CRISPR-Cas9 mediated genome editing ofIFITM3 (as confirmed by next generation sequencing) was achieved by using LentiArray™ Lentiviral sgRNA (Product # A32042, AssayID CRISPR960914_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Fig (a) Western blot analysis of IFITM3 was performed by loading 30 µg of MCF-7 wild type (Lane 1), MCF-7 Cas9 (Lane 2) and MCF-7 Cas9 cells transduced with IFITM3 Lentiviral sgRNA (Lane 3) membrane enriched extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-IFITM3 Polyclonal Antibody (Product # PA5-30382, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:5000 dilution).Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). A loss of signal in sgRNA transduced cells using the LentiArray™ CRISPR product line confirms that antibody is specific toIFITM3 (Fig (b)).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of IFITM3 15 kDa was achieved by transfecting MCF-7 cells with IFITM3 specific siRNAs (Silencer® select Product # S195033, S195034). Western blot analysis (Fig. a) was performed using whole cell lysate from the IFITM3 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with IFITM3 Polyclonal Antibody (Product # PA5-30382, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 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 IFITM3.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti- IFITM3 Polyclonal Antibody (Product # PA5-30382) and a 15 kDa band corresponding to IFITM3 was observed across the cell lines except in Caco-2. Whole cell extracts (30 µg lysate) of NIH/3T3 (Lane 1), THP-1 (Lane 2), PC-3 (Lane 3), MCF-7 (Lane 4) and Caco-2 (Lane 5) were electrophoresed using NuPAGE™ 12% Bis-Tris Protein Gel (Product # NP0341BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti- Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 0.25 µg/ml, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of IFITM3 was performed using 70% confluent log phase MCF7 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with IFITM3 Polyclonal Antibody (Product # PA5-30382) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790, 1:2000 dilution) for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing membrane, lysosomal and endosomal localization. Panel e represents no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: IFITM3 Polyclonal Antibody detects IFITM3 protein at cytosol and membrane on D54MG xenograft by immunohistochemical analysis. Sample: Paraffin-embedded D54MG xenograft. IFITM3 Polyclonal Antibody (Product # PA5-30382) dilution: 1:500. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

PA5-30382

Antibody data