For immunoblotting cell lysates were resolved on SDS polyacrylamide gels and transferred onto Hybond P membranes . Membranes had been blocked with BSA for h then incubated with the following principal antibodies: goat polyclonal anti human actin , mouse monoclonal anti c Myc , mouse monoclonal anti HA ; mouse monoclonal anti actin mouse monoclonal, anti Flag ; rabbit anti PTEN , followed by a horseradish peroxidase conjugated secondary antibody . The proteins had been visualized making use of Western Blotting Luminol Reagents . For immunoprecipitation, cell lysates have been incubated with antibodies against target proteins and protein A Sepharose beads for h at C with gentle agitation. Immunocomplexes bound to protein A Sepharose beads were collected by centrifugation and washed three times in lysis buffer before remaining resolved by sodium dodecyl sulphate polyacrylamide gel electrophoresis . Immunofluorescence microscopy Cos cells have been grown on glass coverslips and transfected through the calcium phosphate strategy. Cells have been grown for h immediately after transfection and fixed in paraformaldehyde in phosphatebuffered saline for min at C and washed with PBS. The cells were permeabilized in . Triton X in PBS for min, washed again in PBS, and incubated in mM glycine in PBS for h at room temperature.
Key and secondary antibodies had been diluted in PBS containing FBS. Cells have been incubated with principal antibodies , rabbit anti Abl , mouse monoclonal anti GM , followed hop over to here by secondary antibodies tetramethyl rhodamine isothiocyanate conjugated anti mouse , Alexa Fluor conjugated anti rabbit, Alexa Fluor conjugated antirabbit and Alexa Fluor conjugated anti mouse for intervals of h having a washing phase in concerning Diamidino phenylindole was utilised to visualize cell nuclei. The coverslips have been mounted on object slides by the use of Fluoromount G . Cells had been photographed by a Hamamatsu ORCA chargecoupled gadget digital camera by using the QED Imaging Strategy application having a Zeiss Axioplan microscope or by a Zeiss Axiocam MRm digital camera working with the AxioVision application with a Zeiss Axiovert CFL microscope . The confocal micrographs in Fig. have been taken with Ultra View Vox confocal microscope and analyzed implementing Volocity program . Lipid binding assay PIP strips had been obtained from Echelon Biosciences .
Dot blot experiments were carried out according to the manufacturer’s protocol. The filter strips had been blocked for min in TBST with fatty acid free of charge BSA and thereafter incubated from the presence from the purified recombinant protein overnight at C. Every single strip was washed three times in TBST Apigenin buffer just before incubating with anti His antibody plus a secondary horseradish peroxidase conjugated anti mouse antibody . Antibody binding was detected by using Luminol Western blotting detection reagents . To make sure lipid binding specificity PIP Array membranes with eight lipids of many concentrations had been applied according the exact same protocol. For pull down assay K human persistent myelogenous leukemia cell line is utilized.