Methods Materials A potent and specific inhibitor of Akt and the Gsk3B inhibitor TWS119 were p53/MDM2 interaction purchased from Calbiochem. Actinomycin D was from Sigma. Nerve growth factor 2. 5 S was obtained from Roche and the iQ SYBR Green Supermix came from Bio Rad. Cell culture The PC12 derived subclones, PC12 and PC12, were isolated as previously described. Cells were grown in DMEM containing 10% horse serum and 5% fetal bovine serum in a 5% CO2 atmosphere. For mainte nance of the subclones, 200 ug/ml of G418 was included in the culture media. Where indicated, 100 ng/ml of NGF was added to the cultures in DMEM containing 1% horse serum and 0. 5% fetal calf serum. SSH Cells were treated with 100 ng/ml of NGF for 2 hrs. Total RNA was prepared with the RNeasy kit and cDNA was synthesized using the SMART PCR cDNA Synthesis Kit.
SSH was carried out with the PCR Select cDNA subtraction kit, as described by the manufacturer. cDNAs synthesized from the total RNA of PC12 and PC12 cells were used as the drivers and tes ters, respectively. Inhibitors,Modulators,Libraries Plasmids encoding subtracted cDNA were Inhibitors,Modulators,Libraries subjected to cycle sequencing using the ABI PRISM 310 genetic analyzer. The sequences obtained were used for sequence alignment with the National Center for Biotechnology Information, GenBank, using a basic Inhibitors,Modulators,Libraries blast search. Semi quantitative and quantitative RT PCR cDNA was synthesized from RNA using the Omniscript RT kit according to the manufacturers protocol. The cDNA mixture was amplified using PCR with specific primers, and the resulting products were separated on 2% agarose gel.
Quantitative PCR was run using SYBR green PCR mastermix and the LightCycler Real time PCR Detection System. The number of amplification steps required to reach the threshold cycle number was computed using LightCycler software 4. 0. To nor malize input cDNA, Gapdh was run separately in all exper iments under the same conditions. The Inhibitors,Modulators,Libraries expression levels of each gene were normalized against Gapdh using the com parative Ct method. Inhibitor treatment Cells were pretreated with the Akt inhibitor AKTi 1/2 or the Gsk3B inhibitor TWS119 for 3 hrs in DMEM and 1% horse serum and 0. 5% fetal calf serum, prior to treatment with 100 ng/ml of NGF for either 10 min or 2 hr. For the transcriptional inhibitor actinomycin D, cells were treated with 100 ng/ml of NGF for 2 hr, followed by 10 uM actinomycin D for 2 hr in the absence of NGF.
Lentivirus vector and infection The lentiviral shRNA vector was constructed Inhibitors,Modulators,Libraries in plasmid pLV TH. Target sequences for knockdown http://www.selleckchem.com/products/lapatinib.html of Syn 1 A nonsi lencing control shRNA . Viral stock was generated by transfecting each lentiviral vector into 293T cells using a standard calcium phosphate precipitation technique. The appropriate lentivi ral vector plasmid and the packaging vector plas mids were co transfected. Viral supernatants were harvested at 48 and 72 hr after transfection and filtered through a 0. 45 um pore size filter.