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. 2018 Dec 17;10(12):521. doi: 10.3390/cancers10120521

Figure 4.

Figure 4

HPV E6 oncoproteins interact with the AT-hook factor AKNA. (A) Purified GST-E6 fusion proteins were incubated for 4 h with a HaCaT cell lysate to allow complex formation. After incubation, GST beads were washed using 0.1% NP-40/PBS solution to wash away unspecific binding. Bound protein was analyzed by western blot using AKNA anti-serum. GST alone was incubated as a binding control. AKNA protein is detected when incubated with GST-E6 proteins. GST-E6 fusion proteins are indicated by the asterisk (*). (B) Anti-E6 (1:25) directed against HPV18 (Santa Cruz Biotechnologies) was used to immunoprecipitate endogenous E6 in HeLa cells, after CBZ treatment to ensure high levels of protein. Anti-AKNA (1:50, Santa Cruz Biotechnologies) and anti-p53 DOI (1:100, Santa Cruz Biotechnologies) and anti-E6 (HPV18) antibodies were incubated overnight with a HeLa cell lysate obtained using RIPA buffer, after that time IgG beads were added to the cell lysate and incubated for 2 h. After several washes bound protein was ascertained by western blot using AKNA anti-serum. Western blot analysis revealed that AKNA is able to complex with E6. p53 was used as an immunoprecipitation control. Longer exposure (lower panel) is showed to demonstrate the presence of AKNA in the input. Note the appearance of AKNA bands detected when p53, AKNA and E6 are immunoprecipitated indicating the in vivo interaction between the analyzed proteins.