AAG has demonstrated promising preclinical activity in vitro in vivo

we examined the potency of MK2206 in regulating the activation state of Akt. HCT116 PTEN cells were treated with MK2206 or LY294002 for 2 h, and then protein lysates were prepared and analyzed by Western blotting. As depicted in Fig. 7A, MK2206 treatment resulted in a dramatic lowering Lenalidomide of degrees of p Akt at both S473 and T308, as well as of the Akt substrate p FoxO1/3a. These effects were more pronounced compared to effects of LY294002 and occurred at significantly lower concentrations. HCT116 PTEN cells were cultured for 3 times in the presence of drug, which led to no overt toxicity and treated with 6 Gy IR in the presence or absence of 2 M MK2206. Cell size was then measured utilizing a Multisizer III. Pharmacological inhibition of Akt did not restore cell size checkpoint control to PTEN deficient cells. To help confirm that Akt was not involved in the radiation induced cell size check-point, HCT116 PTEN cells were transiently transfected using a myr Akt expression construct. Despite expression of p Akt, there clearly was no effect on the integrity of the radiation Gene expression induced cell size check-point. Taken together, these data make sure Akt is not an essential PTEN effector for cell size checkpoint control. Identification of novel putative PTEN effectors via endogenous epitope tagging. We sought to identify novel effectors with this checkpoint, since the ability of PTEN to manage Akt phosphorylation is needless for regulation of the PTEN dependent cell size checkpoint. Specifically, we hypothesized that PTEN interacts with one or several PIP2 or PIP3 regulated proteins as a way to regulate cell size check-point get a handle on. Since recognition of PTEN interacting proteins has demonstrated to be very difficult due, partly, to issues of ectopic over-expression, we developed a new technology, named endogenous epitope tagging. This technique we can efficiently put in a short epitope tag to the allele of genes in cultured human cells to be able to prevent Cediranib ectopic overexpression of epitope labeled transgenes while still using high efficiency affinity reagents for protein complex purification. In a proof of concept experiment for this approach, we described the generation of HCT116 cell lines in which the amino termini of both PTEN alleles were altered with the addition of a 1 FLAG tag. Here, we used these cells to identify novel PTEN interacting proteins. Refinement and mass spectrometric detection of PTENinteracting proteins are described in detail in.. In temporary, protein lysates were prepared from HCT116FLAG PTEN/FLAG PTEN cells and an equal number of negative control HCT116 adult cells and put on a FLAG M2 affinity column, and bound proteins were eluted applying 1 FLAG peptide. The proteins were separated by SDSPAGE, and the protein structure of the eluents was determined using tandem mass spectrometry.