Figure 4: MAN2C1 inhibits PTEN-derived PIP₃ phosphatase activity in both immortalized and primary human prostate epithelial cells.

(a) Immortalized human prostate epithelial BPH-1 cells were infected with MAN2C1 shRNA, PTEN shRNA, their combination, or control shRNA (−) lentivirus as indicated, followed by western blot analysis for PTEN, MAN2C1, phosphorylation of AKT S473 (AKT-P), total AKT and actin. The levels of AKT-P were quantified against AKT and presented under the AKT-P panel as fold changes in comparison with mock-treated cells. (b) PTEN was immunoprecipitated from Ctrl (control) shRNA and MAN2C1 shRNA lentivirus-infected cells (IP:PTEN) and then assayed for PTEN-dependent PIP₃ phosphatase activity in vitro. Experiments were repeated three times and presented as mean±standard deviation. Results were analysed by two-tailed Student's t-test. (c) Human primary epithelial cells were purchased from Sciencell Research labs and infected with a Ctrl shRNA or MAN2C1 shRNA lentivirus. The expression of MAN2C1 (*MAN2C1 band), PTEN and actin was examined by western blot (right panel). PTEN-dependent PIP₃ phosphatase activity was then determined following immunoprecipitation of PTEN (IP:PTEN). Experiments were repeated three times and presented as mean±standard deviation. Results were analysed by two-tailed Student's t-test.