GeneSpring GX 11. 0 software was used to identify statistically significant differences in gene expression between samples. For multiple measurements to detect significantly upregulated and downregulated genes, Inhibitors,Modulators,Libraries the Bonferroni correction was performed by adjust ing the significance level. Fold changes in gene expression, hierarchical clustering, and gene ontology annotations were determined. qRT PCR Total RNA was prepared using the RNeasy Mini Kit at 12, 24, 36 and 48 h after transfection with Tax or the control vector. RT PCR was performed using specific primers and OneStep SYBR Green PCR mix following the manufacturers instructions. The qRT PCR was performed using a 7500 Fast Real time PCR System. All data were nor malized to GAPDH mRNA.
Immunoblot analysis Transfected cells were lysed and proteins were sepa rated on 6%, 10%, or 17% SDS Inhibitors,Modulators,Libraries polyacrylamide gels and then transferred to a PVDF membrane using a Trans blot SD semi dry transfer cell. Following the transfer, the membranes were blocked in 5% non fat dry milk in PBS containing 0. 1% Tween 20 for 1 h and then incubated with a 1,1000 Dacomitinib dilution of primary antibody against Flag, Rb, or actin for 1 h. The membranes were then washed and incubated with anti mouse, anti rabbit, or anti goat horseradish peroxidase conjugated secondary antibodies and developed using the SuperSignal West Pico Chemiluminescent sub strate Kit. Immunofluorescence Cells were seeded onto 22 mm diameter cover slips in 24 well plates and incubated at 37 C for 24 h be fore transfection. Cells were transiently transfected with either a Tax expression vector or a control vector using the Fugene HD reagent.
Twenty four hours later, the cells were washed twice with PBS, fixed in 3. 7% formaldehyde, permeabilized using 0. 2% Triton X 100, and stained with an anti Flag MAb followed by an anti mouse IgG1 antibody conjugated to Alexa Fluor 488 or 494. Subcellular localization was analyzed by confocal laser scanning mi croscopy. Luciferase assay HeLa cells were transfected with 1 ug of Inhibitors,Modulators,Libraries the re porter plasmid, pGV HL21 or pGV, 0. 3 ug of the reference plasmid, pRL SV40, and 0. 5 ug of the Tax expression vector. At 48 h after trans fection, cells were recovered and the activity of firefly and Renilla luciferase was measured in the lysates as previously Inhibitors,Modulators,Libraries described. For each sample, firefly luci ferase activity was normalized by reference to Renilla luciferase activity.
Cell cycle analysis HeLa cells were incubated in a 6 well plate at 37 C for 24 h followed by co transfection for 48 h with 2 ug of the Tax expression vector or the control vector and 0. 2 ug of the pEGFP N1 vector. Cells were collected and washed with PBS without Ca2 and Mg2 and then fixed with 1% paraformaldehyde followed by 70% etha nol. After fixation, cells were washed twice with PBS, treated with 200 ug ml of RNase for 1 h at 37 C, and stained with 50 ug ml of PI.