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. Author manuscript; available in PMC: 2018 Dec 21.
Published in final edited form as: Mol Cell. 2017 Dec 7;68(6):1134–1146.e6. doi: 10.1016/j.molcel.2017.11.006

Figure 2. Pin1 interacts with Ser249-phosphorylated p53-RS.

Figure 2.

A) Pin1 binds to mutant better than wt p53s when overexpressed in cells. Wt p53 and p53-RS plasmids, respectively, were co-introduced with Flag-PIN 1 into H1299 cells. Protein complexes were pulled down and detected by using co-IP-WB or straight WB with indicated antibodies.

B) Endogenous p53-RS and Pin1 interaction. PLC/PRF/5 or HepG2 cells were used for co-IP with the anti-Pin1 antibody followed by WB with indicated antibodies. * indicates the light chain.

C) The schematic of the functional domains and mutated residues of p53K280-4M and p53K280-4MRS..

D) p53K280-4MRS, but not p53K280-4M, interacts with Pin1. p53K280-4M or p53K280-4MRS was co-introduced with Flag-PIN1 to H1299 cells. Protein complexes were pulled down and detected by using co-IP-WB or straight WB with indicated antibodies.

E) Inhibition of phosphatases with Okadaic acid (OA) leads to elevated p53-4MRS-Pin1 association in cells. p53-4MRS was co-introduced with Flag-PIN1 into H1299 cells. Cells were treated by 1 μM or 5 μM OA for 1 h before harvesting and then used for co-IP-WB or straight WB with indicated antibodies.

F) Dephosphorylation of p53-4MRS by CIP reduces its interaction with Pin1. p53-4MRS were co-introduced with Flag-PIN1 into H1299 cells. After harvesting, cells lysates were treated by CIP for 1 h at 37°C, and then used for co-IP-WB or WB with indicated antibodies.

G) CDK4 knockdown reduces p53-4MRS interaction with Pin1. H1299 cells transfected with CDK4 or control SiRNA for 24 h were transfected with combination of plasmids encoding p53-4MRS, Flag-PIN1, harvested for co-IP-WB or straight WB analysis with antibodies as indicated.

H) CDK4 inhibitor PD reduces p53-4MRS interaction with Pin1. H1299 cells were transfected with combinations of plasmids encoding p53-4MRS, Flag-PIN1 treated with PD inhibitor 24 h before harvested for co-IP-WB or straight WB with indicated antibodies.

I) CDK4 increases p53-4MRS interaction with Pin1. H1299 cells were transfected with combination of plasmids encoding p53-4MRS, Flag-PIN1 or CDK4-HA for co-IP-WB or straight WB with indicated antibodies.