701058

antibody from Invitrogen Antibodies

Targeting: AKT1S1 Lobe, MGC2865, PRAS40

Western blot (Data presented on provider website)

Immunocytochemistry (Supportive data in Antibodypedia)

Flow cytometry (Supportive data in Antibodypedia)

Other assay (Supportive data in Antibodypedia)

Antibody Data

Product number

701058

Provider

Invitrogen Antibodies

Product name

Phospho-PRAS40 (Thr246) Recombinant Rabbit Monoclonal Antibody (8H13L13)

Antibody type

Monoclonal

Antigen

Synthetic peptide

Reactivity

Human, Mouse

Host

Rabbit

Isotype

IgG

Antibody clone number

8H13L13

Vial size

100 µg

Concentration

0.5 mg/mL

Storage

Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

References

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)

Immunocytochemistry

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.

Flow cytometry

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.

Other assay

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.