tment is not due to inhibition of Mcl 1 protein synthesis through mTOR signaling

Proteins present particularly in FLAG immunoprecipitates from HCT116FLAG PTEN/FLAG HDAC Inhibitors PTEN cells but not in immunoprecipitates from HCT116 parental cells are listed in Fig. 9B. Not surprisingly, the endogenous FLAG PTEN fusion protein was the most notable differentially immunoprecipitated protein. Other proteins that were present particularly in immunoprecipitates from FLAG PTEN cells involved actin and its remodeling proteins gelsolin and EPLIN. Actin was sufficiently abundant to be visible inside the Coomassie brilliant blue stained gel. Somewhat, gelsolin is regulated by PIP2. Endogenous PTEN colocalizes and interacts with an endogenous PIP2 regulated actin depolymerization complex. To confirm these putative endogenous connections, immunoprecipitation and Western blot analyses were conducted. PTEN was immunoprecipitated from FLAG PTEN cells using FLAG Organism M2 beans, and Western blotting was executed with antibodies for EPLIN, gelsolin, and the three main actin isoforms. As shown in Fig. 10A and 10B, immunoprecipitation of endogenous PTEN generated coimmunoprecipitation of endogenous actin, actin, gelsolin, and EPLIN. Subcellular fractionation experiments demonstrated that the plasma membrane was the only cellular compartment where each one of these proteins was present, suggesting that the interactions were likely to occur in the cell membrane. Subsequent immunoprecipitation and Western blot analyses of sub-cellular fractions proved these interactions occur in the plasma membrane. These studies also demonstrated the connection between PTEN, actin, gelsolin, and EPLIN was insensitive to oxidation state, a regarded regulator of PTEN. The connection between PTEN and actin was more verified by immunoprecipitation /Western Avagacestat blotting using anti PTEN antibodies in genetically unmodified HCT116, LN229, and 293T cells. Next, immunofluorescence was performed to find out whether actin and PTEN colocalize in individual cells. A lentivirus that expresses green fluorescent protein GFP PTEN was developed and used to invade HCT116 PTEN cells. Afflicted cells were then fixed and stained with Alexa conjugated phalloidin, which binds to and spots actin filaments. Cells were then imaged with fluorescence microscopy. As previously described, the most of GFP PTEN was diffusely present in the cytoplasm and the nucleus, having a community present at the plasma membrane. While GFP alone did not colocalize with actin, actin and GFP PTEN colocalized in the plasma membrane. That colocalization was viewed as a delicate but distinctive overlap of GFP and phalloidin staining. These indicators also overlapped with discoloration around the membrane associated actin network. These data are in keeping with the immunoprecipitation and Western blot data indicated in Fig. 10.