Supplementary Figures Supplementary Figure 1. T183 of JNK and WW domain of Pin1 are involved in the interaction. - PowerPoint PPT Presentation
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Supplementary Figures Supplementary Figure 1. T183 of JNK and WW domain of Pin1 are involved in the interaction. (a) HEK 293 cells transfected with plasmids as indicated were treated with 1 mM H 2 O 2 for 1 h or left untreated. Immunoprecipitation and immunoblotting were performed. (b) HEK 293 cells were co-transfected with FLAG-tagged Pin1 WT or two deletion mutants of Pin1, WW or PPIase (amino acid residues 1- 54 or 39-163, respectively) expression plasmids along with the HA-JNK1 plasmid, and exposed to 1 mM H 2 O 2 for 1 h. Immunoprecipitation and immunoblotting were performed. FLAG- tagged Pin1 WW domain was not shown due to its small size. (c) HEK 293 cells were co-transfected with FLAG-tagged Pin1 WT or two point mutants of Pin1 (W34A or C113A) expression plasmids along with the HA-JNK1 plasmid, and exposed to 1 mM H 2 O 2 for 1 h. Immunoprecipitation and immunoblotting were performed. Supplementary Figure 2. H 2 O 2 has no effect on Pin1 activity. Pin1 -/- MEF cells were transiently transfected with FLAG-only or FLAG-Pin1 expression plasmids and then lysates from H 2 O 2 -treated for 1 h or untreated Pin1 -/- MEF cells were immunoprecipitated with anti-FLAG beads. The immunoprecipitates were incubated with active JNK1, c-Jun, and [γ- 32 P] ATP for 10 min at 30°C. Supplementary Figure 3. (a) Pin1does not interact with TCF1. After transfection, HEK 293 cells were treated with 1 mM H 2 O 2 for 1 h. Lysates were immunoprecipitated and immunoblotted with appropriate antibodies. (b) JNK1 phosphorylates Ser-232 of TCF1. Active recombinant JNK1 was incubated with TCF1 (WT, T242A, or S232/T242A) in 20 µl of kinase reaction buffer. The
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Supplementary Figures
Supplementary Figure 1. T183 of JNK and WW domain of Pin1 are involved in the
interaction.
(a) HEK 293 cells transfected with plasmids as indicated were treated with 1 mM H2O2 for 1 h
or left untreated. Immunoprecipitation and immunoblotting were performed. (b) HEK 293 cells
were co-transfected with FLAG-tagged Pin1 WT or two deletion mutants of Pin1, WW or
PPIase (amino acid residues 1-54 or 39-163, respectively) expression plasmids along with the
HA-JNK1 plasmid, and exposed to 1 mM H2O2 for 1 h. Immunoprecipitation and
immunoblotting were performed. FLAG-tagged Pin1 WW domain was not shown due to its
small size. (c) HEK 293 cells were co-transfected with FLAG-tagged Pin1 WT or two point
mutants of Pin1 (W34A or C113A) expression plasmids along with the HA-JNK1 plasmid, and
exposed to 1 mM H2O2 for 1 h. Immunoprecipitation and immunoblotting were performed.
Supplementary Figure 2. H2O2 has no effect on Pin1 activity.
Pin1-/- MEF cells were transiently transfected with FLAG-only or FLAG-Pin1 expression
plasmids and then lysates from H2O2-treated for 1 h or untreated Pin1-/- MEF cells were
immunoprecipitated with anti-FLAG beads. The immunoprecipitates were incubated with
active JNK1, c-Jun, and [γ-32P] ATP for 10 min at 30°C.
Supplementary Figure 3. (a) Pin1does not interact with TCF1. After transfection, HEK 293
cells were treated with 1 mM H2O2 for 1 h. Lysates were immunoprecipitated and
immunoblotted with appropriate antibodies. (b) JNK1 phosphorylates Ser-232 of TCF1.
Active recombinant JNK1 was incubated with TCF1 (WT, T242A, or S232/T242A) in 20 µl
of kinase reaction buffer. The products of kinase reactions were separated by SDS-PAGE. The
gels were dried and exposed to film.
Supplementary Figure 4. The data shown in Figure 4b were quantified as a percentage of
phospho-JNK1.
Supplementary Figure 5. Pin1-induced JNK1 activity is stable.
GST, GST-Pin1, or GST-Pin1 (C113A) proteins were pulled down with GST beads and then
each bead complex was incubated with active JNK1 for 1 h at room temperature. After
removal of GST-fusion proteins by centrifugation, the supernatants were subjected to SDS-
PAGE and then immunoblotted with antibodies as indicated. The same supernatant samples
