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. 2019 Mar 20;116(14):7033–7042. doi: 10.1073/pnas.1819534116

Fig. 1.

Fig. 1.

The HPV16 E10K variant is impaired in PTPN14 degradation but binds RB1 and promotes the expression of E2F-regulated genes. (A) Schematic of protein complexes including HPV E7/RB1 and HPV E7/PTPN14/UBR4. (B) N/Tert-1 keratinocytes were transduced with control and HPV16 E7 retroviruses. Total cell lysates were analyzed by SDS/PAGE/Western blotting and probed with antibodies to PTPN14, RB1, HA, and actin (Upper). HPV16 E7-FlagHA was immunoprecipitated with anti-HA from N/Tert lysates and coimmunoprecipitation of UBR4 and RB1 was assessed by SDS/PAGE/Western blotting (Lower). (C) qRT-PCR for E2F-regulated genes in primary HFK transduced with control and HPV16 E7 retroviruses. Bar graphs display the mean ± SD of two (16E7 Δ21–24) or three (empty vector, 16E7 WT, and 16E7 E10K) independent experiments. Statistical significance was determined by ANOVA followed by multiple t tests with the Holm-Šídák family-wise error rate correction (**P < 0.01; ****P < 0.0001). (D) NTert-1 keratinocytes expressing HPV16 E7 WT were treated with control siRNAs or siRNAs targeting UBR4. Protein levels were assessed by Western blot.