Immunoprecipitation of raptor protein from MCF7 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Raptor (E6O3A) Rabbit mAb. Western blot analysis was performed using Raptor (E6O3A) Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used as the secondary antibody.
Immunoprecipitation of phospho-raptor protein from PANC-1 extracts treated with carbonyl cyanide m-chlorophenylhydrazone (CCCP) (100 μM, 2 hr). Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Phospho-Raptor (Ser792) (E4V6C) Rabbit mAb. Western blot analysis was performed using Phospho-Raptor (Ser792) (E4V6C) Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used as the secondary antibody.
Western blot analysis of extracts from various cell lines using Raptor (E6O3A) Rabbit mAb.
Western blot analysis of extracts from PANC-1 cells, vehicle-treated (-) or treated (+) with combinations of the following treatments as indicated: carbonyl cyanide m-chlorophenylhydrazone (CCCP) (100 μM, 2 hr) and λ phosphatase/calf intestinal alkaline phosphatase (CIP), using Phospho-Raptor (Ser792) (E4V6C) Rabbit mAb (upper) or Raptor (24C12) Rabbit mAb #2280 (lower).
Western blot analysis of extracts from C2C12 cells, vehicle-treated (-) or treated with carbonyl cyanide m-chlorophenylhydrazone (CCCP) (10 μg/mL, 30 min; +), using Phospho-Raptor (Ser792) (E4V6C) Rabbit mAb (upper) or Raptor (24C12) Rabbit mAb #2280 (lower).
PhosphoPlus® Duets from Cell Signaling Technology (CST) provide a means to assess protein activation status. Each Duet contains an activation-state and total protein antibody to your target of interest. These antibodies have been selected from CST's product offering based upon superior performance in specified applications.
The regulatory associated protein of mTOR (Raptor) was identified as an mTOR binding partner that mediates mTOR signaling to downstream targets (1,2). Raptor binds to mTOR substrates, including 4E-BP1 and p70 S6 kinase, through their TOR signaling (TOS) motifs and is required for mTOR-mediated phosphorylation of these substrates (3,4). Binding of the FKBP12-rapamycin complex to mTOR inhibits the mTOR-raptor interaction, suggesting a mechanism for rapamycin's specific inhibition of mTOR signaling (5). This mTOR-raptor interaction and its regulation by nutrients and/or rapamycin is dependent on a protein called GβL (6). GβL is also part of the rapamycin-insensitive complex between mTOR and rictor (rapamycin-insensitive companion of mTOR), and may mediate rictor-mTOR signaling to downstream targets including PKCα (7). Furthermore, the rictor-mTOR complex has been identified as the previously elusive PDK2 responsible for the phosphorylation of Akt/PKB on Ser473, facilitating phosphorylation of Akt/PKB on Thr308 by PDK1 and required for the full activation of Akt/PKB (8).
Recently raptor has been identified as a direct substrate of the AMP-activated protein kinase (AMPK) (9). AMPK phosphorylates raptor on Ser722/Ser792 (9). This phosphorylation is essential for inhibition of the raptor-containing mTOR complex 1 (mTORC1) and induces cell cycle arrest when cells are stressed for energy (9). These findings suggest that raptor is a critical switch that correlates cell cycle progression with energy status.
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