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. 2019 Nov 25;56(1):232–242. doi: 10.3892/ijo.2019.4922

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Copyright: © Xu et al.

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

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Detection of the effects of YAP1/SKP2 axis on mTOR ubiquitination and bladder cancer progression. (A and B) HT-1376 and J82 cells were trans-fected with siRNAs-SKP2; then, the cells were harvested and subjected to RT-PCR and western blot assays to determine the knockdown efficiency of SKP2 (^**P<0.01, ^***P<0.001). (C) Immunoprecipitation assay was used to assess the effects of the YAP1/SKP2 axis on mTOR protein expression and ubiquitination. (D and E) Western blotting was performed to test the expression and phosphorylation of mTOR, p-mTOR, p-eIF4E, elF4E, p-rpS6 and rpS6 following cell transfection. (F and G) The effects of the YAP1/SKP2 axis on cell proliferation were determined by CCK-8 analysis. Detection of the effects of YAP1/SKP2 axis on mTOR ubiquitination and bladder cancer progression. (H) The effects of the YAP1/SKP2 axis on cell apoptosis were assessed by flow cytometry analysis. (I and J) Western blotting was performed to detect the expression of apoptosis-related proteins, such as caspase 3/9 and cleaved-caspase3/9 after 48 h of cell transfection (D-J, OE-YAP1 + si-NC group or OE-NC + si-SKP2 group vs. OE-NC + si-NC group, ^*P<0.05, ^**P<0.01, ^***P<0.001; OE-YAP1 + si-SKP2 group vs. OE-YAP1 + si-NC group, ^#P<0.05, ^##P<0.01; NC, negative control). YAP1, Yes-associated protein 1; SKP2, S-phase kinase-associated protein 2; RT-PCR, reverse transcription-polymerase chain reaction; eIF, eukaryotic translation initiation factor; rpS6, ribosomal protein s6.

Detection of the effects of YAP1/SKP2 axis on mTOR ubiquitination and bladder cancer progression. (A and B) HT-1376 and J82 cells were trans-fected with siRNAs-SKP2; then, the cells were harvested and subjected to RT-PCR and western blot assays to determine the knockdown efficiency of SKP2 (^**P<0.01, ^***P<0.001). (C) Immunoprecipitation assay was used to assess the effects of the YAP1/SKP2 axis on mTOR protein expression and ubiquitination. (D and E) Western blotting was performed to test the expression and phosphorylation of mTOR, p-mTOR, p-eIF4E, elF4E, p-rpS6 and rpS6 following cell transfection. (F and G) The effects of the YAP1/SKP2 axis on cell proliferation were determined by CCK-8 analysis. Detection of the effects of YAP1/SKP2 axis on mTOR ubiquitination and bladder cancer progression. (H) The effects of the YAP1/SKP2 axis on cell apoptosis were assessed by flow cytometry analysis. (I and J) Western blotting was performed to detect the expression of apoptosis-related proteins, such as caspase 3/9 and cleaved-caspase3/9 after 48 h of cell transfection (D-J, OE-YAP1 + si-NC group or OE-NC + si-SKP2 group vs. OE-NC + si-NC group, ^*P<0.05, ^**P<0.01, ^***P<0.001; OE-YAP1 + si-SKP2 group vs. OE-YAP1 + si-NC group, ^#P<0.05, ^##P<0.01; NC, negative control). YAP1, Yes-associated protein 1; SKP2, S-phase kinase-associated protein 2; RT-PCR, reverse transcription-polymerase chain reaction; eIF, eukaryotic translation initiation factor; rpS6, ribosomal protein s6.