Immunofluorescence analysis of Phospho-mTOR / FRAP pSer2448 was done on 70% confluent log phase A549 cells treated with serum starvation followed by 200ng of EGF for 10 minutes. 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 of Phospho-mTOR/FRAP pSer2448 Rabbit Polyclonal Antibody (441125G) at 2ug/ml in 0.1% BSA and incubated for 3 hours at room temperature 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 (S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d is a merged image showing cytoplasmic localization. Panel e is untreated cell with no signal. Panel f is a no primary antibody control. The images were captured at 60X magnification.
|Tested species reactivity||Human, Mouse|
|Published species reactivity||Human|
|Host / Isotype||Rabbit / IgG|
|Immunogen||The antiserum was produced against a chemically synthesized phosphopeptide derived from the region of human mTOR that contains serine 2448. The sequence is conserved in human, mouse and rat.|
|Purification||Antigen affinity chromatography|
|Storage buffer||Dulbecco's PBS, pH 7.3, with 1mg/ml BSA, 50% glycerol|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||3-5 µg/10^6 cells|
|Immunofluorescence (IF)||Assay Dependent|
|Western Blot (WB)||Assay Dependent|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that plays a key role in cell growth, cell proliferation, and protein synthesis. mTOR mediates phosphoinositide 3-kinase and Akt/PKB signaling, resulting in phosphorylation of 4EBP1, and initiation of mRNA translation. A second pathway involves regulation of ribosomal S6 kinase, which affects ribosome biogenesis and translation elongation.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
Phase II study of the oral MEK inhibitor selumetinib in advanced acute myelogenous leukemia: a University of Chicago phase II consortium trial.
44-1125G was used in flow cytometry to determine the clinical relevance of the RAS/RAF/MEK/ERK pathway in patients with acute myelogenous leukemia
|Jain N,Curran E,Iyengar NM,Diaz-Flores E,Kunnavakkam R,Popplewell L,Kirschbaum MH,Karrison T,Erba HP,Green M,Poire X,Koval G,Shannon K,Reddy PL,Joseph L,Atallah EL,Dy P,Thomas SP,Smith SE,Doyle LA,Stadler WM,Larson RA,Stock W,Odenike O||Clinical cancer research : an official journal of the American Association for Cancer Research (20:490)||2014|
|Human||Not Cited||Identification of S6 kinase 1 as a novel mammalian target of rapamycin (mTOR)-phosphorylating kinase.||Holz MK,Blenis J||The Journal of biological chemistry (280:26089)||2005|