Western blot analysis of extracts from serum-starved HeLa cells, untreated or treated with IGF-1 (50 ng/ml, 30 minutes) and λ phosphatase, using Phospho-Rictor (Thr1135) (D30A3) Rabbit mAb (upper) or Rictor (53A2) Rabbit mAb #2114 (lower).
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Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
Protocol Id: 10
Phospho-Rictor (Thr1135) (D30A3) Rabbit mAb detects endogenous levels of rictor protein only when phosphorylated at Thr1135.Species Reactivity:
Human, Mouse, Monkey
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to the sequence surrounding Thr1135 of human Rictor protein.
Cell growth is a fundamental biological process whereby cells accumulate mass and increase in size. The mammalian TOR (mTOR) pathway regulates growth by coordinating energy and nutrient signals with growth factor-derived signals (1). mTOR is a large protein kinase with two different complexes. One complex contains mTOR, GβL and raptor, which is a target of rapamycin. The other complex, insensitive to rapamycin, includes mTOR, GβL, Sin1, and rictor (1). The mTOR-rictor complex phosphorylates Ser473 of Akt/PKB in vitro (2). This phosphorylation is essential for full Akt/PKB activation. Furthermore, an siRNA knockdown of rictor inhibits Ser473 phosphorylation in 3T3-L1 adipocytes (3). This complex has also been shown to phosphorylate the rapamycin-resistant mutants of S6K1, another effector of mTOR (4).
Phosphorylation of Thr1135 on rictor was identified at Cell Signaling Technology (CST) using PhosphoScan®, CST's LC-MS/MS platform for phosphorylation site discovery (5). Additional research indicates that rictor is phosphorylated at Thr1135 by p70 S6K, which negatively regulates mTORC2 protein complex as part of a negative feedback mechanism controlling Akt activity (6-8).
Explore pathways + proteins related to this product.
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