Fig 2: Rap1GAP is degraded by the UPP in HPV16/18 positive cervical cancer cells. Total cell lysates (as indicated) were used to detect the expression of Rap1GAP in the cells (input) and subjected to immunoprecipitation with an anti-Rap1GAP antibody. The pulled-down proteins were analyzed using immunoblotting with the anti-ubiquitin antibody to detect the interaction between Rap1GAP and ubiquitin. To avoid the disturbance bands at 55 kD and 25 kD formed by the antibody used for pull-down in co-IP, the anti-Rap1GAP antibody was from rabbit and the anti-ubiquitin antibody was from mouse in co-IP (see “Methods” section)

Fig 3: Knocking down E6AP increase the levels of Rap1GAP in HPV-positive cervical cancer cells. HeLa, SiHa and C33A cells were transfected with siRNA for E6AP, and the levels of E6AP were detected by western blotting to check the efficiency of knocking down. Then, the levels of Rap1GAP and p53 were detected by western blotting after silencing E6AP. *p < 0.05

Fig 4: Rap1GAP interacts with E6AP in HPV16/18 positive cervical cancer cells. Total cell lysates from HeLa, SiHa and C33A cells were used to detect the expression of Rap1GAP and E6AP in the cells (input). Then, the lysates were subjected to immunoprecipitation with an anti-Rap1GAP antibody. The pulled-down proteins were analyzed using immunoblotting. Interaction between Rap1GAP and E6AP was detected with an anti-E6AP antibody, and the immunoprecipitation efficiency of Rap1GAP was verified with an anti-Rap1GAP antibody

Fig 5: Rap1GAP is degraded by the autophagy pathway. Cells, as indicated, were treated with rapamycin for 72 h to induce cell autophagy. The cell lysates were used to check the autophagy markers p62 and LC3 II/I to assess the levels of autophagy. Then, the levels of Rap1GAP were detected by western blotting to check the effect of autophagy on the degradation of Rap1GAP