Western blot analysis of extracts from 293 cells (starved for 30 hours), untreated or treated with 20% FBS for 30 minutes, using Phospho-mTOR (Ser2481) Antibody (upper) or control mTOR Antibody #2972 (lower).
Flow cytometric analysis of 293 cells using mTOR (7C10) Rabbit mAb (blue) compared to a nonspecific negative control antibody (red).
Western blot analysis of extracts from various cell lines, using Raptor (24C12) Rabbit mAb.
Western blot analysis of extracts from various cell lines, using Rictor (53A2) Rabbit mAb.
Western blot analysis of extracts from GβL wild-type (+/+) and knockout (-/-) mouse embryonic fibroblast cells using GβL (86B8) Rabbit mAb. (provided by David Sabatini Lab at the Whitehead Institute for Biomedical Research).
Confocal immunofluorescent analysis of HeLa cells, rapamycin-treated (#9904, 10 nM for 2 hours, left), insulin-treated (150 nM for 6 minutes, middle) or insulin- and λ-phosphatase-treated (right), using Phospho-mTOR (Ser2448) (D9C2) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin. Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
Confocal immunofluorescent analysis of mouse embryonic fibroblast (MEF) cells using mTOR (7C10) Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Western blot analysis of extracts from various cell lines, using GβL (86B8) Rabbit mAb.
Western blot analysis of extracts from serum-starved NIH/3T3 cells, untreated or insulin-treated (150 nM, 5 minutes), alone or in combination with λ-phosphatase, using Phospho-mTOR (Ser2448) (D9C2) XP® Rabbit mAb (upper) or mTOR (7C10) Rabbit mAb #2983.
Immunohistochemical analysis of paraffin-embedded human breast carcinoma, showing cytoplasmic localization using mTOR (7C10) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human lung carcinoma, using mTOR (7C10) Rabbit mAb in the presence of control peptide (left) or mTOR Blocking Peptide #1072 (right).
Immunohistochemical analysis of paraffin-embedded mouse brain using mTOR (7C10) Rabbit mAb.
Western blot analysis of extracts from 293, A431, COS, C6, and C2C12 cells, using mTOR (7C10) Rabbit mAb.
Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Fluorescein Conjugate) #6201 (-) or SignalSilence® mTOR siRNA II (+), using mTOR (7C10) Rabbit mAb #2983 and α-Tubulin (11H10) Rabbit mAb #2125. mTOR (7C10) Rabbit mAb confirms silencing of mTOR expression, while the α-Tubulin (11H10) Rabbit mAb is used to control for loading and specificity of mTOR siRNA.
|Phospho-mTOR (Ser2481) Antibody 2974||20 µl||
||H M R Mk||289||Rabbit|
|mTOR (7C10) Rabbit mAb 2983||20 µl||
||H M R Mk||289||Rabbit IgG|
|Raptor (24C12) Rabbit mAb 2280||20 µl||
||H M R Mk||150||Rabbit|
|Rictor (53A2) Rabbit mAb 2114||20 µl||
||H M R Mk||200||Rabbit IgG|
|GβL (86B8) Rabbit mAb 3274||20 µl||
||H M R Mk||37||Rabbit IgG|
|Phospho-mTOR (Ser2448) (D9C2) XP® Rabbit mAb 5536||20 µl||
||H M R Mk||289||Rabbit IgG|
|Anti-rabbit IgG, HRP-linked Antibody 7074||100 µl||
The mTOR Pathway Antibody Sampler Kit contains reagents to investigate the control of protein translation, cell growth, and proliferation through mTOR signaling within cells. The kit contains enough primary and secondary antibodies to perform two Western blot experiments per primary antibody.
Each total antibody in the mTOR Pathway Sampler Kit recognizes only its specific target. Each phospho-specific antibody detects the intended target only when phosphorylated at the indicated site.
Polyclonal antibody is produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Ser2481 of human mTOR. Polyclonal antibodies are purified by protein A and peptide affinity chromatography. Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser2448 of human mTOR, Gln1681 of human Rictor, Gln210 of human GβL and human Raptor.
The mammalian target of rapamycin (mTOR, FRAP, RAFT) is a Ser/Thr protein kinase (1-3) that functions as an ATP and amino acid sensor to balance nutrient availability and cell growth (4,5). When sufficient nutrients are available, mTOR responds to a phosphatidic acid-mediated signal to transmit a positive signal to p70 S6 kinase and participate in the inactivation of the eIF4E inhibitor, 4E-BP1 (6). These events result in the translation of specific mRNA subpopulations. mTOR is phosphorylated at Ser2448 via the PI3 kinase/Akt signaling pathway and autophosphorylated at Ser2481 (7,8). mTOR plays a key role in cell growth and homeostasis and may be abnormally regulated in tumors. For these reasons, mTOR is currently under investigation as a potential target for anti-cancer therapy (9).
The regulatory associated protein of mTOR (Raptor) interacts with mTOR to mediate mTOR signaling to downstream targets (10,11). Raptor binds to mTOR substrates, such as 4E-BP1 and p70 S6 kinase, through their TOR signaling (TOS) motifs and is required for mTOR-mediated substrate phosphorylation (12,13). Binding of the FKBP12-rapamycin complex to mTOR inhibits mTOR-raptor interaction, which suggests a mechanism for the inhibition of mTOR signaling by rapamycin (14). This mTOR-raptor interaction and its regulation by nutrients and/or rapamycin is dependent on a protein called GβL (15). GβL is part of the rapamycin-insensitive complex between mTOR and rictor (rapamycin-insensitive companion of mTOR) and may mediate rictor-mTOR signaling to PKCα and other downstream targets (16). The rictor-mTOR complex has been identified as the previously elusive PDK2 responsible for the phosphorylation of Akt/PKB at Ser473, which is required for PDK1 phosphorylation of Akt/PKB at Thr308 and full activation of Akt/PKB (17).
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