Figure 4. p73 is recruited to ΔNp73 in promoter LMP-1 expressing cells.

(A) SaOS-2 cells were transfected with different pcDNA3 constructs in the indicated combinations. After 36 hours, ChIP was performed using an anti HA-tag antibody and followed by real-time PCR, using primers flanking the p53-RE within the ΔNp73 promoter. Simultaneously, 1/10 of the total chromatin was processed. The values in the histogram were obtained by dividing for each sample the amount of ΔNp73 promoter, which is bound by p73-HA and p53-HA for the total amount of ΔNp73 promoter present in the input. The reported values are the mean of three independent experiments. (B and C) RPMI-pLXSN (pLXSN) and RPMI-pLXSN-LMP-1 (LMP-1) were fixed and processed for ChIP, or collected to perform DNA-pull down. (B) ChIP was carried out using an anti p73 antibody. The real time PCR and the quantification of the % of p73 binding to the p73-RE within the ΔNp73 promoter, was performed as explained in the legend of Figure 4A. The data are the mean of three independent experiments. The difference in the p73 levels recruited to ΔNp73α promoter in presence and absence of LMP-1 is statistically significant ((p = 0.000254) (C) After the DNA pull-down assay the levels of p53 and p73 proteins binding to biotinylated DNA probe were analysed by immunoblotting. (D) LCLs were transfected with scrambled RNA and siRNA for p53 (sip53) or transduced with lentivirus carrying two different OmicsLink p73 shRNAs (shp73-1, shp73-2) and scramble shRNA. Thirty-six hours after transfection or one-week post lentivirus transduction cells were collected and processed for RNA extraction and cDNA synthesis. The levels of p53, p73 and ΔNp73α transcript were determined by quantitative RT-PCR. The data are the mean of three independent experiments. The difference in mRNA ΔNp73α levels in scramble or sip53 transfected cells was not significant, while it was statistically significant between scramble and shp73-1 (p value = 0.0022) and scramble and shp73-2 (p value = 0.005).