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. Author manuscript; available in PMC: 2016 Apr 1.
Published in final edited form as: Neurobiol Dis. 2015 Jan 6;76:13–23. doi: 10.1016/j.nbd.2014.12.027

Fig. 2.

Fig. 2

Pin1 is oxidized on Cys113 in vitro and in cells.

A) Validation of oxyPin1 antibodies in vitro. Purified recombinant Pin1 was treated with H2O2 or vehicle, followed by immunoblot analysis with oxyPin1 or Pin1 antibodies.

B) Validation of Pin1 oxidation on Cys 113 in cells. Cells were treated with H2O2 and followed by pull-down with biotin-pTide beads before subjecting to immunoblotting with oxyPin1, Pin1 and PTEN antibodies.

C) Pin1 is oxidized on Cys113. Cells were co-transfected with Flag-Pin1 or its mutants and then treated with H2O2. Cell extracts were pulled down with biotin-pTide beads and immunoblotted by oxyPin1 and Flag antibodies.

D) Pin1 is oxidized on Cys113 after diamine treatment. Cells were co-transfected with Flag-Pin1 or its mutants and then treated with 250 µM Diamine for 15 min. Oxidized Pin1 were labeled with biotin-maleimide, and cell extracts were purified with streptavidin-sepharose beads, and immunoblotted by oxyPin1 and Flag antibodies.