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. Author manuscript; available in PMC: 2014 Jul 1.
Published in final edited form as: Cancer Res. 2013 Apr 30;73(13):10.1158/0008-5472.CAN-12-4360. doi: 10.1158/0008-5472.CAN-12-4360

Figure 3.

Figure 3

SENP1 binds to Pin1 and promotes Pin1-mediated cyclin D1 activation.

A,. 293T cells were co-transfected with Flag-Pin1, GFP-SUMO1 and HA-SENP1, HA-SENP2 or HA-SENP3 constructs and subjected to immunoprecipitation, followed by immunoblotting. B, Top, 293T cells were transfected with Pin1 with different amounts of HA-SENP1 or HA-SENP2 constructs, followed by Flag IP and immunoblotting. Bottom, The same amount of SENP1 or SENP2 with SUMOylated Pin1 were incubated in a deSUMOylation reaction buffer, followed by immunoblotting. C, Top, 293T cells were co-transfected with Flag-SENP1 or Flag-SENP2 and Pin1 constructs and then subjected to immunoprecipitation with anti-Flag, followed by immunoblotting with Pin1 Ab. Bottom, Glutathione-agarose beads containing GST or GST-Pin1 were incubated with cell extracts expressing Flag-SENP1 or Flag-SENP2. Proteins pulled down by GST beads were subjected to immunoblotting. D, HeLa cells were transfected with SENP1 or SENP2, and Pin1, or vector control, along with cyclin D1 promoter reporter construct, followed by assaying the luciferase and pRL-TK Renilla luciferase activity as control. Arrowhead: SUMO1-modified Pin1, arrow: SUMO1-unmodified Pin1 and asterisk: IgG.