Western blot analysis of extracts from MCF7 cells, untreated or H2O2-treated (2.5 mM, 30 min), using Phospho-MOB1 (Thr12) (D2E3) Rabbit mAb (upper) and MOB1 Antibody #3863 (lower).
Western blot analysis of extracts from Ramos cells, untreated or λ phosphatase-treated, using Phospho-MOB1 (Thr12) (D2E3) Rabbit mAb (upper) and MOB1 Antibody #3863 (lower).
|REACTIVITY||H M R Mk|
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-MOB1 (Thr12) (D2E3) Rabbit mAb recognizes endogenous levels of MOB1 protein only when phosphorylated at Thr12.Species Reactivity:
Human, Mouse, Rat, MonkeySpecies predicted to react based on 100% sequence homology:
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr12 of human MOB1 protein.
MOB1 was first identified in yeast as a protein that binds to Mps with essential roles in the completion of mitosis and the maintenance of ploidy (1). Its Drosophila and mammalian homologs, Mats and MOB1, respectively, are involved in the Hippo signaling tumor suppressor pathway, which plays a critical role in organ size regulation and which has been implicated in cancer development (2-5). There are two MOB1 proteins in humans, MOB1α and MOB1β, that are encoded by two different genes but which have greater than 95% amino acid sequence identity (6). Both forms bind to members of the nuclear Dbf2-related (NDR) kinases, such as LATS1/2 and NDR1/2, thereby stimulating kinase activity (7-9). This binding is promoted by the phosphorylation of MOB1 at several threonine residues by MST1 and/or MST2 (5,10).
Phosphorylation at Thr12 by MST1/2 stabilizes MOB1, enhancing its binding and regulation of LATS1 (5). The resultant increase in LATS1 kinase activity promotes inhibitory phosphorylation of the transcriptional co-activators YAP and TAZ (11,12), leading to changes in the expression of genes involved in cell cycle progression (13).
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