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. 2019 Dec 12;94(1):e00663-19. doi: 10.1128/JVI.00663-19

FIG 5.

FIG 5

HPV regulates p27 protein expression by targeting NHERF-2. (A) HA-tagged NHERF-2 was overexpressed in HEK-293 cells, alone or in combination with HPV-16 E6 or -18 E6 or with their respective mutants, HPV-16 E6ΔPBM or HPV-18 E6ΔPBM, as indicated. As a negative control, HEK-293 cells were transfected with the EV. β-Galactosidase (LacZ) was used as an internal standard to monitor transfection efficiency, and γ-tubulin was used as a loading control. After 24 h of transfection, cells were harvested, and lysates were prepared and analyzed by Western blotting for the expression of p27 and NHERF-2 proteins using anti-p27 and anti-NHERF-2 antibodies. Relative densitometries for p27 and NHERF-2 under various transfection conditions are shown below. (B) Cell lysates from the experiment shown in panel A were used to check the expression levels of HA–NHERF-2, cyclin D1, and CDK4 proteins. Relative densitometries for cyclin D1 and CDK4 under various transfection conditions are shown below. β-Galactosidase (LacZ) was used as an internal standard to monitor transfection efficiency, and γ-tubulin was used as a loading control. (C) HEK-293 cells were cotransfected with the indicated plasmids alone, with the EV, or in combination with the control luciferase siRNA (siLuc) or NHERF-2 siRNA (siNHERF-2). Twenty-four hours after transfection, cells were harvested, and whole-cell lysates were prepared and analyzed by Western blotting using various antibodies as indicated. β-Galactosidase (LacZ) was used as an internal standard to monitor transfection efficiency, while β-actin was used as a loading control. Relative densitometries for p27 and HA–NHERF-2 under various transfection conditions are shown below. One representative of at least 3 independent Western blots is shown. The data are expressed as fold change relative to γ-tubulin (A and B) or to β-actin (C). In each case, the mean values and SEM of the results of 3 independent experiments are shown. *, P < 0.05; **, P < 0.01; ns, not statistically significant. (D and E) HeLa and CaSki cells were transfected with siRNA directed against luciferase (siLuc), E6/E7 (siE6/E7), E6AP (siE6AP), and NHERF-2 (siNHERF-2), alone or in combination. After 72 h, cells were harvested, and the levels of NHERF-2, p53, p27, and the α-actinin loading control were detected by Western blotting. (F) NHERF-2, p53, and p27 protein levels were analyzed by Western blotting in cell lysates from C33-A (HPV-negative), HeLa (HPV-18-positive), and CaSki and SiHa (both HPV-16-positive) cells. β-Actin was used as a loading control, and in each case, one representative of at least three independent Western blots is shown.