Fig. 3

USP13 maintains PTEN expression through direct interaction. a. Expression of USP13, PTEN as well as phosphor-AKT and AKT were detected in USP13 knocked down 5637 and UM-UC-3 cells by western blot analysis, the band intensities were quantitated in b. c. agomir of miR-130b/301b was transfected into 5637 and UM-UC-3 cells, and USP13 expression was subsequently rescued. Western blotting analysis was performed to evaluate the expression of USP13 and PTEN, the band intensities were quantitated in d. e. miR-130b/301b was knocked down in 5637 and UM-UC-3 cells, and expression of USP13 and PTEN was detected by western blotting analysis, the band intensities were quantitated in f. g. NF-kB p65 was overexpressed in 5637 cells, and then agomir of miR-130b, miR-301b or pLvx-USP13 was transfected into the NF-kB p65 overexpressed cells, respectively. Expression of phosphor-p65, USP13 and PTEN was measured by western blotting analysis, the band intensities were quantitated in h. i. 293 T cells were transfected with Flag-tagged USP13 or empty vector for 24 h, then followed by TNF-a or relative vehicle treatment for 12 h. Lysates were subjected to immunoprecipitation with anti-Flag M2 beads. Bound proteins were analyzed by western blotting with USP13 or PTEN antibodies, the band intensities were quantitated in j. The internal control genes were GAPDH for western blot analysis and β-actin for real-time PCR analysis. The gels were run under the same experimental conditions. The band intensities were calculated by Image J 1.46r software, and the ratio of target gene to GAPDH was used to conduct the statistical analysis. *P < 0.05 and **P < 0.01, as determined by Student’s T-test