The usage of fluorescently marked proteins and functional scientific studies showthat this routinely led to reasonably even expression of target proteins in effectively more than 95% on the cultured U87MG cells as described previously. In most experiments in U87MG cells, baculoviruses were utilised to express PTEN at similar levels to endogenous levels in other cultured cells, even though, in protein screening library stability experiments, levels were five ten instances greater in order to aid the detection of 35S labelled PTEN in immunoprecipitates. Antibodies and Western blotting Phospho particular antibodies against PTEN phospho Thr366 and PTEN phospho Ser370 had been raised employing the phosphopeptides TSVT?PDV and TPDVS?DNE respectively. These peptides, as well as a PTEN Nterminal peptide MTAIIKEIVSRNKRRY, were synthesized by Dr Graham Bloomberg and had been injected into sheep at Diagnostics Scotland. Sheep had been also immunized with total length hexahistidine tagged PTEN protein expressed and purified from bacteria. Antibodies were purified from serum by affinity for the immunized peptide or protein.
All blotting experiments 17,20 lyase inhibtors employing affinity purified phospho distinct antibodies integrated co incubation using the corresponding dephosphopeptide to block non phospho precise immunoreactivity. Mouse monoclonal and rabbit polyclonal antibodies to PTEN were purchased from Santa Cruz Biotechnology and Biosource respectively.
Polyclonal antibodies to phospho PTEN and phospho Ser473 Akt/PKB had been purchased from Cell Signaling Technologies. A rabbit polyclonal antibody raised against PTEN phospho Ser385 was bought from Biosource. To prepare cellular samples for protein gel electrophoresis, the following cell lysis buffer was employed: 25 mM Hepes, 150 mM NaCl, 1% Triton X one hundred, 0.1% 2 mercaptoethanol, 1 mM EDTA, 1 mM EGTA, ten mM glycerophosphate, 50 mM sodium fluoride, ten g/ml leupeptin, one hundred M PMSF and 1 mM benzamidine. Proteins had been separated by Web page making use of pre cast four 12% gradient gels and blotted on to PVDF membranes. Most reagents for electrophoresis and blotting were purchased from Invitrogen, and standard producers, protocolswere followed. Immunoprecipitation of PTEN utilised the A2B1 monoclonal antibody from Santa Cruz Biotechnology, pre conjugated with agarose beads. The assay of immunoprecipitated Akt/PKB activity followed procedures published previously. Quantification was performed utilizing AIDA image analysis software. Inside the situation of data in Supplementary Figures 2 and three, the scanned and presented images derived from processed film were employed. In the situation of data from Figure 4 and Supplementary Figure 4, blots were analysed straight working with a CCD camera.