- 701058 - Invitrogen Antibodies AKT1S1 antibody

Submitted references Secondary Metabolites from the Culture of the Marine-derived Fungus Paradendryphiella salina PC 362H and Evaluation of the Anticancer Activity of Its Metabolite Hyalodendrin.

Dezaire A, Marchand CH, Vallet M, Ferrand N, Chaouch S, Mouray E, Larsen AK, Sabbah M, Lemaire SD, Prado S, Escargueil AE
Marine drugs 2020 Apr 3;18(4)

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of Phospho-AKT1S1 pThr246 in whole cell extracts of HepG2 cells treated with PDGF (100 ng/mL, 15 min) using a Phospho-AKT1S1 pThr246 recombinant rabbit monoclonal antibody (Product # 701058) at a dilution of 2.5 µg/mL. To confirm specificity, competition was performed by preincubation with the phosphopeptide to inhibit antibody binding (lane 1). Results show a band at ~40 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of PRAS40 (pT246) was performed by loading whole cell extracts of 30 µg of untreated NIH/3T3 (lane 1) and NIH/3T3 treated with PDGF (100 ng/mL for 15 minutes) lysates (lane 2) 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. PRAS40 (pT246) was detected at ~36 kDa using PRAS40 (pT246) Recombinant Rabbit Monoclonal Antibody (Product # 701058) at a 1:1000 dilution in 2.5% skim milk at 4°C overnight on a rocking platform. To confirm specificity, competition was performed with the phosphopeptide (10 µg/mL) (lane 3). Detection was performed using an HRP-conjugated Goat anti-Rabbit secondary antibody (Product # G-21234) at a 1:5000 dilution and chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of Phospho-AKT1S1 pThr246 in whole cell extracts of HepG2 cells treated with PDGF (100 ng/mL, 15 min) using a Phospho-AKT1S1 pThr246 recombinant rabbit monoclonal antibody (Product # 701058) at a dilution of 2.5 µg/mL. To confirm specificity, competition was performed by preincubation with the phosphopeptide to inhibit antibody binding (lane 1). Results show a band at ~40 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of PRAS40 (pT246) was done by treating serum starved U-87 MG cells with 20 ng/mL PDGF for 15 minutes. 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 PRAS40 (pT246) Recombinant Rabbit Monoclonal Antibody (Product # 701058) at a dilution of 1:400 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 and nuclear localization. Panel e shows competition with the phospho- PRAS40 (pT246) peptide. The images were captured using a Nikon microscope at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of PRAS40 (pT246) was performed on U-87 MG cells treated with PDGF (100 ng/ml, 15 minutes). 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 PRAS40 (pT246) recombinant rabbit monoclonal antibody (Product # 701058, red histogram) or with rabbit isotype control (pink histogram) at a dilution of 1:250 in 2.5% BSA. After incubation at room temperature for 3 hours, the cells were labeled with Alexa Fluor¨ 488 goat anti-Rabbit Secondary antibody (Product # 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)
Main image
Experimental details: Figure 4 Intracellular signaling pathways modulated in MCF7-Sh-WISP2 cells exposed to 3 . ( A ) MCF7-Sh-WISP2 cells were treated or not with 2.8 uM of 3 for 48 h. The cells were lysed and the protein extracts were subjected to a protein array analysis. The table chart on the right of the blot indicates the individual proteins tested here. Results are representative of two individual experiments. ( B ) MCF7-Sh-WISP2 cells were treated or not with 2.4 uM of 3 for 72 h. The cells were then lysed and the antigens revealed by immunolabelling using antibodies directed against p53, phospho-p53 (Serine 15), PRAS40 and phospho-PRAS40 (Threonine 246). beta-Actin immunoblot served as a loading control. ( C ) Western blots from three independent experiments were quantified by densitometry and values expressed as (phospho-p53/total p53) or (phospho-PRAS40/total PRAS40) ratios. The statistical analysis of experimental data was performed using Student's paired t-test comparing the hyalodendrin-treated samples with the vehicle control. Results are expressed as means +- standard deviation (SD), * p < 0.05.

701058

Antibody data