Western blots for phosphorylated molecules were utilised as surrogate markers for pathway activation. Phosphorylation of PKB in melanoma and melanocytes In order to set up if PIK3CA, PTEN, NRAS and BRAF mutations resulted in constitutive activation of the downstream signalling pathways, we measured PKB activation by western blotting for phosphorylation at two online websites, Ser473 and Thr308. Equal quantities of protein from NZM cell lines have been loaded onto the same gel, but for clarity, western blots were segmented to display results for person NZM cell lines. In melanocytes, phosphor ylation of PKB on each Ser473 and Thr308 was strongly serum dependent although many of the NZM cell lines on this review showed serum independent phosphorylation.
PKB was phosphorylated independently of serum in the mTORC2 dependent Ser473 web site in many of the cell lines, despite the fact that NZM46 and NZM3 surprisingly had quite low levels of phosphorylation even while in the presence of serum In contrast, phosphorylation with the PIP3 PDK1 dependent Thr308 web-site tended to be low in the serum starved state in most cell lines selleckchem GSK2118436 and increased with serum The notable exceptions have been cell lines NZM12, NZM40 and NZM52 which have paratively substantial Thr308 phosphorylation in serum starved cells. Phosphorylation of Thr308 during the NZM40 and NZM52 cell lines may possibly be explained by the activat ing PIK3CA mutation in these cells. These two cell lines also possess a very low level of total PKB suggesting some feedback regulation of PKB gene expression in these cells. In support of this, NZM46, which also features a PIK3CA mutation also has really high PTEN levels which could clarify the reduced Thr308 phosphorylation in these cells and the greater ranges of total PKB pared to NZM40 and NZM52, as PIP3 levels might be predicted for being low regardless of the PIK3CA mutation.
NZM46 demonstrates suppression of phosphoryla tion by serum in the Thr308 web page Phosphorylation of ponents of the mTOR pathway in melanoma cells and melanocytes Activation of ponents of your protein translation machinery has become observed inside a big JNK-IN-8 1410880-22-6 percentage of melanomas and is predictive of a poor prognosis The PI3K signalling pathway can regulate protein trans lation machinery via mTORC1 and subsequent activation of p70S6K and phosphorylation of ribosomal protein S6 Hence we next established the phosphorylation standing of p70S6K The p70S6K was strongly expressed in all cell lines at the same time as in ordinary melanocytes however the pattern of phosphoryla tion of p70S6K and p85S6K at Thr389 did not correlate with all the phosphorylation standing of PKB nor did it corre late with genotypes In melanocytes, the observed phosphorylation of Ser235 236 was serum dependent whereas Ser240 244 site, and that is phosphory lated by p70S6K, was phosphorylated even while in the absence of serum.