Figure 3: MAN2C1 inhibits the PIP3 phosphatase activity of PTEN.
From: α-Mannosidase 2C1 attenuates PTEN function in prostate cancer cells
(a) The indicated recombinant GST fusion proteins (GST, GST-PTEN/PTEN and GST-MAN2C1/MAN2C1) were purified from E. coli and 0.5 μg of individual recombinant proteins were analysed. #Indicates the molecular mass of the respective recombinant proteins (top panel). Western blotting was performed on 5 ng of individual recombinant proteins using anti-MAN2C1 or anti-PTEN antibodies (bottom panel). (b) The indicated doses of GST or GST-MAN2C1 were assayed for their effect on 0.3 μg GST-PTEN associated PIP3 phosphatase activity. Experiments were repeated three times. Means±standard derivations are shown. *P<0.05 (two-tailed Student's t-test). (c–e) A quantity of 5 μg PTEN (P5) and 0, 5, 10 and 20 μg MAN2C1 (M0P5, M5P5, M10P5, M20P5) (c), C-MAN236 (C0P5, C5P5, C10P5, C20P5) (d) or Cat-MAN (CA0P5, CA5P5, CA10P5, CA20P5) (e) were coexpressed in 293T cells and IP with indicated antibody and 60 μl agarose G matrix. A volume of 15 μl matrix was used to perform the lipid phosphatase assay. 5 μg PTEN(C124S) (C124S) and 20 μg MAN2C1 (M20P0) (c), C-MAN236 (C20P0) (d) or Cat-MAN (CA20P0) (e) were expressed in 293T cells and IP accordingly. IP proteins were detected by western blotting. The assay was performed four times. Values are presented as mean±standard deviation. Coimmunoprecipitated MAN2C1 and its mutants (c–e) were quantified and presented as fold changes underneath the individual panels. (f) DU145 cells were transfected with control (Ctrl) or MAN2C1 siRNA lentivirus and selected in puromycin for 48 h to ensure 100% infection. Cells were then analysed for the expression of PTEN, MAN2C1 and actin (right panel). MAN2C1 levels were quantified and presented as fold changes in comparison with Ctrl siRNA cells underneath the individual panels. PTEN was IP from Ctrl and MAN2C1 siRNA-treated cells and assayed in vitro for its PIP3 phosphatase activity. Experiments were repeated three times and presented as means±standard deviations. Results were analysed by two-tailed Student's t-test.
