Fig 4.

Negative regulation of IRS-1 stability via C1-Ten PTPase activity. (A) Ponceau S staining revealed the quantity, integrity, and purity of the full-length C1-Ten protein. (B) Time-dependent PTPase activity of C1-Ten. The malachite green assay was performed with 200 ng of C1-Ten and 400 μM EEEEpYEEEE peptide (n = 3). (C) Cysteine-based PTPase activity of C1-Ten. C1-Ten CS and PTEN were used as the negative and positive controls, respectively. Error bars indicate the SEMs of four independent experiments, each performed in triplicate. (D) Vanadate-mediated inhibition of C1-Ten PTPase activity. Error bars indicate the SEMs of three independent experiments, each performed in duplicate. (E) CHX chase. HEK293 cells were transfected with vector (Vec), Flag–C1-Ten WT, or Flag–C1-Ten CS. After 48 h, CHX (20 μg/ml) and 10 nM insulin (Ins) were added and chased for the times indicated, followed by immunoblot analysis (left) and graphical analysis (right). (F) HEK293 cells were transfected with vector or Flag-PTEN. After 48 h, CHX and insulin were added and chased, followed by immunoblot analysis (left) and graphical analysis (right). *, P < 0.05; **, P < 0.01; ***, P < 0.001.