- AHO1382 - Invitrogen Antibodies EIF4EBP1 antibody

Submitted references Inhibition of the autophagic protein ULK1 attenuates axonal degeneration in vitro and in vivo, enhances translation, and modulates splicing.

Vahsen BF, Ribas VT, Sundermeyer J, Boecker A, Dambeck V, Lenz C, Shomroni O, Caldi Gomes L, Tatenhorst L, Barski E, Roser AE, Michel U, Urlaub H, Salinas G, Bähr M, Koch JC, Lingor P
Cell death and differentiation 2020 Oct;27(10):2810-2827

Polycystin-1 affects cancer cell behaviour and interacts with mTOR and Jak signalling pathways in cancer cell lines.

Papavassiliou KA, Zoi I, Gargalionis AN, Koutsilieris M
Journal of cellular and molecular medicine 2019 Sep;23(9):6215-6227

Effect and molecular mechanism of mTOR inhibitor rapamycin on temozolomide-induced autophagic death of U251 glioma cells.

Li B, Zhou C, Yi L, Xu L, Xu M
Oncology letters 2018 Feb;15(2):2477-2484

Induction of genuine autophagy by cationic lipids in mammalian cells.

Man N, Chen Y, Zheng F, Zhou W, Wen LP
Autophagy 2010 May;6(4):449-54

Butyrate-rich colonic microenvironment is a relevant selection factor for metabolically adapted tumor cells.

Serpa J, Caiado F, Carvalho T, Torre C, Gonçalves LG, Casalou C, Lamosa P, Rodrigues M, Zhu Z, Lam EW, Dias S
The Journal of biological chemistry 2010 Dec 10;285(50):39211-23

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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) and U-87 MG (Lane 5). The blot was probed with Anti-4EBP1 antibody (Product # AHO1382, 1:500 dilution) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 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)
Main image
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 # AHO1382, 1:500 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 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: 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 (554R16)(Product # AHO1382) using 1:1,000 dilution and Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 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)
Main image
Experimental details: Immunofluorescent analysis of 4E-BP1 was done on 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with 4E-BP1 Mouse Monoclonal Antibody (Product # AHO1382) at 1:250 dilution in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor 488 Rabbit Anti-Mouse IgG Secondary Antibody (Product # A-11059) at a dilution of 1:400 for 30 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 Alexa Fluor 594 Phalloidin (Product # A12381). Panel d is a merged image showing nuclear and perinuclear localization. Panel e shows no primary antibody control. The images were captured at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
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 Monoclonal Antibody (Product # AHO1382) at 1:250 dilution in 0.1% BSA and incubated overnight at 4 degree and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) 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)
Main image
Experimental details: Flow cytometry analysis of 4E-BP1 was done on HEK-293 cells. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Tritonª X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with 4E-BP1 Mouse Monoclonal Antibody (AHO1382, red histogram) or with mouse isotype control (pink histogram) at 3-5 µg/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor¨ 488 Rabbit Anti-Mouse Secondary Antibody (A11059) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune¨ Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 6 Both AAV.ULK1.DN and the ULK1 inhibitor SBI-0206965 enhance mTOR activation. Lysates were obtained from E18 rat cortical neurons on DIV 8 after transduction with AAV.ULK1.DN or AAV.CTRL. Alternatively, cells were treated with the ULK1 inhibitor SBI-0206965 (SBI, 5 muM) or DMSO as control for 30 min before lysis. RAP addition of rapamycin (750 nM) 24 h before lysis. a - h Top: Representative immunoblots of mTOR, p-mTOR, S6, p-S6, 4E-BP1, p-4E-BP1, AMPK, p-AMPK, and the corresponding bands of the loading controls tubulin or GAPDH after transduction with AAV.ULK1.DN or AAV.CTRL are shown. Bottom: Quantifications of the band intensities of mTOR, p-mTOR, p-S6, AMPK (all n = 6 independent cultures), p-4E-BP1 ( n = 5 independent cultures), p-AMPK ( n = 4-5 independent cultures), 4E-BP1 ( n = 4 independent cultures), and S6 ( n = 3 independent cultures) normalized to tubulin or GAPDH as loading controls. CTR control, RAP treated with rapamycin. i-p Top: Representative immunoblots of mTOR, p-mTOR, S6, p-S6, 4E-BP1, p-4E-BP1, AMPK, p-AMPK, and the corresponding bands of the loading control GAPDH after treatment with SBI or DMSO as control are shown. Bottom: Quantifications of the band intensities of mTOR, p-mTOR, p-S6, AMPK, p-AMPK, 4E-BP1, p-4E-BP1 (all n = 5 independent cultures), and S6 ( n = 4 independent cultures) normalized to GAPDH as loading control. CTR control, RAP treated with rapamycin. Data are presented as single data points and means +- SEM. * P < 0.05, ** P < 0.0

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4 Effect of Pkd1 silencing on the mTOR pathway in cancer cell lines. (A-E) Western blot analysis showing the effect of Pkd1 silencing on the phosphorylation of mTOR pathway components in MCF7, PC3, A549, HT29 and GOS3 cells. (F-J) Quantitative data showing the effect of Pkd1 silencing on the phosphorylation of mTOR pathway components in MCF7, PC3, A549, HT29 and GOS3 cells. Bars represent means +- SD. * P < 0.05, ** P < 0.01 versus non-target. siPKD1 represents cancer cells transfected with siRNA targeting the Pkd1 mRNA; non-target represents cancer cells transfected with a non-targeting siRNA; mock represents cancer cells transfected with only transfection reagents (without siRNA)

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5 Effect of PC1 inhibition on the mTOR pathway in cancer cell lines. (A-E) Western blot analysis showing the effect of IgPKD1 on the phosphorylation of mTOR pathway components in MCF7, PC3, A549, HT29 and GOS3 cells. (F-J) Quantitative data showing the effect of IgPKD1 on the phosphorylation of mTOR pathway components in MCF7, PC3, A549, HT29 and GOS3 cells. Bars represent means +- SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 versus mock. Mock represents cells that have been incubated for 3 h with non-immune rabbit serum; 1 h, 3 h, and 6 h represent time points of cell harvesting after 3 h incubation of cancer cells with the IgPKD1 antibody

AHO1382

Antibody data