Figure 6.

E6AP/p53 physical interaction in feline oral SCC cells expressing FcaPV-2 E6. (a) Cell lysates from SCCF2 and SCCF3 were incubated with anti-p53 antibody for immunoprecipitation (IP) or with non-specific antibody (IgG) as control. E6AP and ubiquitin were revealed by WB upon incubation of the membrane with the specific antibodies. The membrane was stripped and blotted with anti-p53 antibody to confirm the occurrence of the IP. SCCF2 cell lysate was loaded to check for molecular weight of the IP bands (control). An aliquot of IP samples was kept before IP as input and analysed by WB for β-actin to ensure that equal amounts of proteins were used for each cell line. IP and control boxes are cut from the same gel at different exposure times, and properly aligned according to the molecular weight standard loaded onto the gel. Full length blots with molecular markers are shown in Supplementary information (Supplemental Fig. S12). Representative gels out of two independent experiments, demonstrating co-IP of E6AP with p53 in both cell lines and higher p53 poly-ubiquitination in SCCF3 are shown. E6AP was immunoprecipitated by incubation of SCCF2 (b) and SCCF3 (c) cell lysates with the specific antibody and the presence of p53 in the immune-complex as well as in input and supernatants (SN) revealed by WB using enhanced ECL reagents. The specificity of the co-IP was checked by parallel incubation with beads alone (Beads) and non-specific antibodies (IgG). The same membrane was blotted for E6AP to confirm the IP. Input samples were loaded onto the same gel to check for the right molecular weight of the co-IP bands and calculate the percentage of p53 bound to E6AP. IP and input boxes are cut from the same gel at the same exposure time and properly aligned according to the molecular weight standard loaded onto the gel. Representative gels demonstrating co-IP of p53 with E6AP are shown.