- MA5-15005 - Invitrogen Antibodies EIF4EBP1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of 4EBP1 was achieved by transfecting A-431 cells with 4EBP1 specific validated siRNAs (Silencer® select Product # S223471, S2234472). Western blot analysis (Fig. a) was performed using whole cell extracts from the 4EBP1 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with 4EBP1 Monoclonal Antibody (Product # MA5-15005, 1:500 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/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 4EBP1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on whole cell extracts of A-431 (Lane 1), HEK-293 (Lane 2), HeLa (Lane 3), Hep G2 (Lane 4), U-87 MG (Lane 5) and NIH/3T3 (Lane 6). The blot was probed with Anti-4EBP1 antibody (Product # MA5-15005, 1:500 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 17 kDa band corresponding to 4EBP1 was observed across the cell lines tested.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockout of EIF4EBP1 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, AssayID CRISPR648136_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of EIF4EBP1 was performed by loading 30 µg of HeLa wild type (Lane 1), HeLa CAS9 (Lane 2) and HeLa EIF4EBP1 KO (Lane 3) whole cell extracts. The samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). 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-4EBP1 Monoclonal Antibody (E.992.6) (Product # MA5-15005) using 1:1,000 dilution and Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4,000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to EIF4EBP1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of 4EBP1 was performed using 70% confluent log phase A-431 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with 4EBP1 Rabbit Monoclonal Antibody (Product # MA5-15005) at 1:250 dilution in 0.1% BSA and incubated overnight at 4 degree 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 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 nuclear and cytoplasmic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of 4E-BP1 in HeLa cells using a 4E-BP1 monoclonal antibody (Product # MA5-15005) (green). Actin filaments are labeled with a fluorescent red phalloidin. DNA is labeled using a fluorescent blue dye.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical analysis of 4E-BP1 in paraffin-embedded human lung carcinoma using a 4E-BP1 monoclonal antibody (Product # MA5-15005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometric analysis of 4E-BP1 in COS cells using a 4E-BP1 monoclonal antibody (Product # MA5-15005) (blue) compared to a nonspecific negative control antibody (red).

MA5-15005