Figure 2.

Co-immunoprecipitation (co-IP) of E6AP with FcaPV-2 E6 and p53 in CRFKE6. (a) Cell lysates from CRFKE6 and CRFKpCEFL were incubated with anti-HA antibody for immunoprecipitation (IP) of HA-tagged FcaPV-2 E6 or with non-specific antibody (IgG) as control. E6AP was revealed by WB upon incubation with the specific antibody. The membrane was stripped and blotted with anti-HA antibody to confirm the occurrence of the IP. CRFKE6 cell lysate was loaded to check for molecular weight of the IP bands (control). An aliquot of samples was kept before IP as input and analysed by WB for E6AP, HAE6 and for β-actin to ensure that equal amount of proteins were used for each cell line. Boxes of IP are cut from the same gel at the same exposure times, control square is cut at a different exposure time 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. S11). A representative co-IP out of two independent experiments, demonstrating physical interaction of E6AP and FcaPV-2 E6, is shown. (b) p53 was immunoprecipitated by incubation of cell lysates with specific antibody and the presence of E6AP and ubiquitin in the immunocomplex revealed by WB. The same membrane was blotted for p53 to confirm the IP. The molecular weight of IP bands was checked by loading whole cell lysate along with IP samples. Input were analysed for β-actin as above. A representative co-IP out of two independent experiments, demonstrating interaction of E6AP with p53 and high poly-ubiquitination of p53 in CRFKE6, is shown.