01% Tween 20 and probed with the following primary antibodies in 3% NFM in TBS T overnight at 4 C. rabbit anti cyclin A1, mouse anti cyclin A2, mouse protein inhibitors anti cdc2, rabbit anti CDK2, rabbit anti p53, mouse anti Hsp70, mouse anti p130/Rb2 full length, rabbit anti ser ine 952 phosphorylated p130/Rb2, rabbit anti serine 2 phosphorylated RNA poly merase II, rabbit anti serine 5 phosphorylated RNA polymerase II, mouse anti a tubu lin, and mouse anti ser139 phos phorylated histone gH2AX. Membranes were washed for 15 minutes in TBS T and then incubated for 1 hour with either goat anti mouse or mouse anti rabbit horseradish peroxidase conju gated IgG at a dilution of 1 10,000 in 3% NFM in TBS T. This was followed by 15 minutes of wash in TBS T and enhanced chemiluminescence according to the manufacturers instructions.

All western blot Inhibitors,Modulators,Libraries images included in article are representative of at least three consecutive indepen dent experiments. Immunostaining Following respective drug treatments, cells grown Inhibitors,Modulators,Libraries directly on sterilized glass coverslips were fixed and per meabilized for 10 minutes in 70% cold methanol, immunostained and analyzed Inhibitors,Modulators,Libraries as previously described. Flow cytometry Cells were collected, after respective drug treatments, washed, resuspended in 1 ml of PBS and fixed and permeabilized for at least 10 minutes in 70% cold ethanol. After fixation cells were pelleted, washed 3 times with PBS, re suspended into a primary antibody solution and incu bated Inhibitors,Modulators,Libraries on ice for 15 minutes. Cells were then pelleted, washed 3 times with PBS, re suspended into FITC con jugated secondary antibody solution and incubated for 15 minutes on ice protected from the light.

Cells were washed 3 times in PBS and re sus pended in propidium iodide staining Inhibitors,Modulators,Libraries solution, 10 ug/ml propidium iodide and 25 ug/ml DNase free RNase A diluted in PBS. Cells were incu bated at 37 C for a minimum of 30 minutes protected from light and analyzed immediately by flow cytometry utilizing an Epics XL MCL BeckmanCoulter. Graphs represent average fluorescence intensity or average percentage of cells found in cell cycle phase over three consecutive inde pendent experiments. Reverse Transcriptase PCR and Real time Total RNA from cell lines was extracted using the High Pure RNA Isolation Kit following the manufac turers instruction.

cDNA was synthesized from 1 ug of total RNA by using random hexamers as primers and moloney murine leukemia virus reverse transcriptase according the manufacturers protocol in a final volume of 20 ul. As a control for genomic contamination a reverse transcription http://www.selleckchem.com/products/Enzastaurin.html reaction was carried out without the addition of the reverse transcriptase. After cDNA synthesis, sam ples were diluted 1 10 and 4 ul was used in each real time polymerase chain reaction. cDNA was amplified using species specific intragenic primers for CCNA1, CCNA2, CCNB1, CCND3, CCNE1, TP53 and GAPDH genes.