FIG 9.

Effect of Rint1 and Rint1-N overexpression on E2-dependent virus replication. C33a cells were transfected with HPV16 Ori (p16OriM), HPV16 E2, and HA-E1 either alone or in combination with increasing amounts of an HA-Rint1 expression plasmid (A and B) or a FLAG–Rint1-N expression plasmid (C and D). (A and C) Cell lysates were analyzed by Western blotting using HPV16 E2-specific, HA-specific (to detect E1 and the full-length Rint1 protein) or FLAG-specific (to detect the FLAG–Rint1-N protein) antibodies. Western blots were stripped and reprobed with a β-actin-specific antibody as a loading control. (B and D) Transient DNA replication of p16OriM was measured by qPCR and compared to a standard curve generated by serial dilution of the purified plasmid. The data shown are means and standard deviations from technical replicates (**, P < 0.01; ***, P < 0.001). The results are the means and standard errors from a single experiment performed in triplicate and are representative of data from at least three independent repetitions of each experiment.