- 700392 - Invitrogen Antibodies AKT1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of AKT (pS473) was performed by loading 20 µg of NIH/3T3 (lane1), NIH/3T3 treated for 10 minutes with 25 ng/mL of PDGF (lane2) and U-87 MG (lane3) cell lysates using Novex®NuPAGE®4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (Product # LC5800), and iBlot® Dry Blotting System (Product # IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk for 1 hour at room temperature. AKT (pS473) was detected at ~55 kDa using AKT (pS473) Recombinant Rabbit Monoclonal Antibody (Product # 700392) at 1 µg-3 µg/mL in 2.5 % skim milk at 4°C overnight on a rocking platform. To confirm specificity, competition was performed with phosphopeptide (10 µg/mL) as shown in the corresponding blot on right. Goat anti-Rabbit IgG-HRP Secondary Antibody (Product # G-21234) at 1:5000 dilution was used and chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of Phospho-AKT pSer473 in untreated NIH-3T3 cell lysate and PDGF-treated NIH-3T3 (lane 2) using a Phospho-AKT pSer473 recombinant rabbit monoclonal antibody (Product # 700392) at a dilution of 0.1 µg/mL.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of Phospho-AKT pSer473 in mouse fibroblasts cells treated with 10 µg/mL Insulin (top right) or untreated (top left) using a Phospho-AKT pSer473 recombinant rabbit monoclonal antibody (Product # 700392) at a dilution of 5 µg/mL followed by detection using an Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody at a dilution of 1:1000 and nuclei staining using Hoechst (blue). Signal is knocked down after incubation with the phosphopeptide used as an immunogen (bottom left) but not with the non-phosphopeptide (bottom right).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of AKT (pS473) was done on 70% confluent log phase U-87 MG cells. The cells were fixed with 4% paraformaldehyde for 15 minutes; permeabilized with 0.25% Triton X-100 for 10 minutes followed by blocking with 5% BSA for 1 hour at room temperature. The cells were incubated with AKT (pS473) Recombinant Rabbit Monoclonal Antibody (Product # 700392) at 1 µg-2 µg in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor® 488 Goat anti-Rabbit IgG Secondary Antibody (Product # A-11008) 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 cytoplasmic localization of AKT (pS473). Panel e shows competition with AKT (pS473) peptide. The images were captured at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of Phospho-AKT pSer473 in paraffin-embedded human esophagus carninoma using a Phospho-AKT pSer473 monoclonal antibody (Product # 700392) at a dilution of 0.5 µg/mL. Tissues were pretreated with EDTA and staining was visualized using DAB. Images were taken at a magnification of 20x. Results show nuclear and cytoplasmic staining in tumor cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry analysis of AKT [pS473] was done on U-87 MG 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 1 hour at room temperature. Cells were labeled with ABfinityª AKT [pS473] Recombinant Rabbit Monoclonal Antibody (700392, red histogram) or with rabbit isotype control (pink histogram) at 2.5 µg/million cells in 2.5% BSA. After incubation at room temperature for 2-3 hours, the cells were labeled with Alexa Fluor¨ 488 Goat Anti-Rabbit Secondary Antibody (A11008) 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)
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Experimental details: Flow cytometry analysis of Phospho-AKT pSer473 in Jurkat cells incubated with 50uM LY294002 for 1hr (red) or untreated (green) using a Phospho-AKT pSer473 recombinant rabbit monoclonal antibody (Product # 700392) at a dilution of 1 µg. Cells were fixed and permeabilized using FIX & PERM (Product # GAS-004) reagent, and detection was performed using an Alexa Fluor 488 goat anti-rabbit IgG compared to a control without primary antibody (blue).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of Phospho-AKT pSer473 in Jurkat cells incubated with 50uM LY294002 for 1hr (red) or untreated (green) using a Phospho-AKT pSer473 recombinant rabbit monoclonal antibody (Product # 700392) at a dilution of 1 µg. Cells were fixed and permeabilized using FIX & PERM (Product # GAS-004) reagent, and detection was performed using an Alexa Fluor 488 goat anti-rabbit IgG compared to a control without primary antibody (blue).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5. VEGFRs in PI 3-kinase and integrin activation. (A) VEGFR knockdown. HUVECs transfected with the indicated siRNAs for 72 h were analyzed for VEGFR expression by immunoblotting. Three independent experiments gave similar results. (B) PI3K signaling. HUVECs transfected as in A were subjected to shear stress and p85 was immunoprecipitated. Samples were then analyzed by immunoblotting and densitometry as in Fig 2 C . Values are means +- SEM, n = 4. (C) Akt signaling. siRNA-treated HUVECs were subjected to laminar shear stress for the indicated times and lysates were analyzed by immunoblotting with the indicated antibodies to determine Akt activity. Results were quantified by densitometry, with actin serving as a loading control. Values are means +- SEM (error bars), n = 3. *, P < 0.05 relative to unstimulated cells by two-way ANOVA. IB, immunoblotting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig 4 Deguelin downregulates AKT/NFkappaB mediated prosurvival signals in CLL cells. (A) PBMCs from 3 CLL patients were treated for 24h with 0, 10, or 100 muM deguelin. Viability was evaluated by flow cytometry after Annexin V/IP staining. Figure shows immunoreactive bands for several deguelin target molecules and beta-actin in western blots from a representative sample. Numbers indicate the signal intensity of beta-actin-normalized bands from drug treated samples compared to the untreated ones (Control). (B) PBMCs from two CLL patients were treated with 0, 10, or 100 muM deguelin or 1 mug/ml fludarabine and co-cultured with Ltk - 24h. Cell viability assays and western blot from a representative sample were done as in (A). (C) PBMCs from 2 CLL patients were co-cultured with Ltk - or 3T3-CD40L cells and p-AKT expression was evaluated in CLL cells by intracellular staining and flow cytometry at different time points. Histograms show the stains in one of the samples (solid lines from left to right: p-AKT staining at 0, 24, 48 and 120h; dotted lines from left to right: unstained control (samples without primary antibody plus secondary antibody) and isotype control. The lower graph shows the differences in the Mean Fluorescence Intensity (MFI, in channel numbers) of p-AKT compared to the unstained control in both samples (bars show mean and SD). The dotted horizontal line indicates the mean difference in MFI (10 +- 5) between the isotype controls and corresponding unstained contro

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 2 Chronic preconception male ethanol exposure exerts sex-specific effects on offspring metabolic function. a Fasting blood glucose levels compared between preconception treatments ( n = 15 males, 15 females). b Fasting insulin levels compared between preconception treatments ( n = nine males, nine females). c - f Glucose tolerance (GTT) and insulin tolerance (ITT) tests in the offspring of ethanol-exposed and control males ( n = 15 males, 15 females). Organ weights of g male and h female offspring compared between the two preconception treatment groups ( n = nine males, nine females). i Representative immunoblot comparing total and phosphorylated AKT (Ser473) between male and female offspring sired by ethanol-exposed and control males ( n = six males and six females). j Densitometry analysis of immunoblots comparing total and phosphorylated AKT ( n = six males and six females). Data analyzed using either an unpaired t test or a two-way ANOVA followed by Sidak post hoc analysis. Error bars represent SEM * p < 0.05 and ** p < 0.01 (comparisons between alcohol and control preconception treatments)

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 2 Immunohistochemical pattern in clear cell renal cell carcinomas displaying nuclear and/or cytoplasmic and/or membranous staining. a Snail, b Twist, c ZEB1, d beta-catenin, e E-cadherin, f Vimentin, g PTEN, h p110alpha, i p-Akt, j SETD2

700392

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