In vitro p53 degradation and ubiquitination assays and mrna quantitation by quantitative pcr

Supplementary information: detailed protocols for in vitro p53 degradation and ubiquitination assays and mRNA quantitation by quantitative PCR.
In vitro p53 degradation and ubiquitination assays

p53, E6 or F47R proteins were produced and labeled with ³⁵S Met in rabbit reticulocytes lysates by using the Quick T7 coupled transcription/translation kit (Promega). MBP-E6 and MBP-E6 F47R fusions proteins were produced as previously described (Zanier et al., 2007) and adjusted at 20 M concentrations in buffer A (Phosphate 20 mM pH 6.8 , NaCl 200 mM, DTT 2 mM, 10mM maltose). For the degradation assay, 5 l of labeled E6 or F47R and 2 l of labeled p53 were mixed in Tris pH 7.5 25 mM, NaCl 100 mM, DTT 3 mM and incubated at 25°C for 2 hours. For figure 1D, E6/p53 mixture was incubated at 25°C for two hours with 2 l of Buffer A or 2 l of MBP-E6 or MBP- E6 F47R protein (or dilutions in buffer A). Proteins were loaded on a 12% SDS polyacrylamide gel and visualized by autoradiography.

For the ubiquitination assay, 2.5 l of in-vitro translated ³⁵S labeled E6 and p53 proteins were incubated in 30-l reaction mixture containing Tris pH 7.5 25 mM, NaCl 100 mM, DTT 3 mM, 9 l of rabbit reticulocytes lysates, and 4 mM of ATP-S (Sigma) for 30 min at 25°C. The reaction mixture was then incubated with anti-p53 monoclonal antibodies (Ab-6, Calbiochem). The proteins were collected on protein-G sepharose beads (GE Healthcare), washed with LSAB (Tris pH 8 100 mM, NaCl 100 mM, NP40 1%, DTT 2mM, anti-proteases cocktail) and released from the beads by boiling in Tris pH 6.8 60 mM, SDS 2%, glycerol 10%, -mercaptoethanol 1% for 7 min. The beads were pelleted and the supernatant was diluted into 400 l of LSAB buffer and immunoprecipitations were performed with anti-ubiquitin antiserum (Sigma). Proteins were collected on protein G-sepharose beads, resolved on a SDS-12% polyacrylamide gel and visualized by autoradiography.
mRNA quantitation by quantitative PCR

Cells (100-mm dishes) were transfected with pNKA, pNKA-E6 or pNKA-E6 F47R plasmids. Twenty-four hours after transfection, cells were trypsinated, diluted to 1/6, split and selected with 0.4 g/ml of puromycin. At day 6 post-transfection, total RNA was prepared using RNAeasy Plus Mini kit (Qiagen) by following the manufacturer’s instructions. Total RNA (400 ng) was reverse-transcribed into cDNA using random primers p(dN)₆ with Superscript II Reverse Transcriptase according to the manufacturer’s instructions (Invitrogen). Each real-time PCR reaction was performed in technical duplicate with cDNA from transfected cells by using Quantitec SYBR Green PCR kit (Qiagen) according to the manufacturer’s instructions. The 18E6 primers (sens 5’-TGGTGTATAGAGACAGTATACCCCA, reverse 5’-GCCTCTATAGTGCCCAGCTATGT) were designed to amplify a 251 pb fragment within the full-length 18E6/E7 cDNA (corresponding to sequence 265-516 in HPV18 genome, the E6 sens primer hybridized with a sequence in the 18E6 intron). The 18E7 primers (sens 5’-CCGAGCACGACAGGAACGACT and reverse 5’-TCGTTTCTTCCTCTGAGTCGCTT) were designed to amplify a 174 pb fragment present both in 18E6*/E7 cDNA and full-length 18E6/E7 cDNA as previously described (Bernard et al., 2003) (corresponding to sequence 531-705 within the HPV18 genome). The hPBGD (human phosphobilinogen deaminase) gene was used as an internal control to normalize the variations in mRNA amounts (sens 5’-TGCACGATCCCGAGAC and reverse 5’-CGTGGAATGTTACGAGC). The efficiency of each primers set was determined by standard curve experiments : 95% for E6 primers set, 100% for hPBGD primers set and 100% for E7 primers set. For E6 reaction, the PCR cycling program consisted of denaturing at 95°C for 15 min and 58 cycles at 94°C for 15 s, annealing at 58°C for 25 s, and elongation at 72°C for 25 s and contained cDNA and 1M of final concentration of E6 primers. For E7 and hPBGD reactions, the PCR cycling program consisted of denaturing at 95°C for 15 min and 55 cycles at 94°C for 15 s, annealing at 56°C for 20 s and elongation at 72°C for 20 s and contained cDNA and 0.5 M of final concentration of E7 primers or hPBGD primers. Gene expression levels were shown relative to hPBGD mRNA levels. The relative quantification of target transcripts was calculated by the two Ct method (Livak and Schmittgen, 2001).