FIG 5.

Reduced expression of cystatin E/M in HPV 16 E7 immortalized HEK cells. (A) Western blot analysis reveals expression of cystatin E/M in normal human epidermal keratinocytes (HEK cells) and HEK cells immortalized with the HPV 16 E6 gene or E7 gene or with the E6 gene plus the E7 gene. However, reduced expression was seen in the HEK/HPV 16 E7 cell line in comparison to the normal cell line or the HEK/HPV 16 E6 cell line. (B) Immunofluorescence staining confirms reduced expression in HEK/HPV 16 E7 cells (×100 magnification). The inset for a single cell shows uniform expression in HEK/HPV 16 E6 cells and reduced punctuated staining in HEK/HPV 16 E7 cells. (C) Western blot analysis of the supernatants (100 μl) again shows cystatin E/M expression in the immortalized HEK cells with reduced expression in HEK/HPV 16 E7cells. (D) Hybridization with the FLAG antibody shows cystatin E/M expression in the HeLa cells transfected with the FLAG tag cystatin E/M plasmid. Expression was not seen in HeLa cells transfected with the E6 gene or the E7 gene alone. (E) Hybridization of HA (HPV 16 E6 or E7 gene tag)-immunoprecipitated samples with the FLAG (cystatin E/M tag) antibody shows the presence of cystatin E/M protein in cells cotransfected with the HPV 16 E7 gene, pointing to an interaction between HPV 16 E7 and cystatin E/M proteins. (F) Hybridization of anti-cystatin E/M antibody-immunoprecipitated lysates with the antiubiquitin (Ubq) antibody shows higher-intensity hybridization of high-molecular-mass proteins in HPV 16 E7- and cystatin E/M-cotransfected HeLa cells, indicating cystatin E/M degradation by HPV 16 E7. This interpretation is also supported by the absence of a visible cystatin E/M band in the cell-free supernatants of HPV 16 E7- and cystatin E/M-cotransfected cells. β-Tubulin and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) were used as the input controls.