Fig 2: Enriched novel environment exposure and seizure induce Kv4.2 phosphorylation at a Pin1 binding site.a Enriched novel environment (EE, 1 h) induces phosphorylation of Kv4.2 at Thr607 but not Thr602 in mouse hippocampus. n = 5 in each group. T-test, *p < 0.05. b Kainic acid-induced seizure (25 mg/kg, i.p., 15 min) induces phosphorylation of Kv4.2 at Thr607 but not Thr602 in mouse hippocampus. n = 4 in each group. T-test, ***p < 0.001. c PTZ-induced seizure (50 mg/kg, i.p., 15 min) induces phosphorylation of Kv4.2 at Thr607 and Thr602 in mouse hippocampus. n = 4 in each group. *p < 0.05, T-test, **p < 0.01. d PTZ-induced seizure increases Pin1 binding to Kv4.2. GST or GST-Pin1-linked beads were incubated with brain lysates from mice subjected to saline or PTZ administration. n = 5 in each group. T-test, **p < 0.01. e Mouse brain lysates from WT mice w or w/o PTZ administration (50 mg/kg, i.p., 15 min) were incubated with excess anti-Kv4.2, anti-Kv4.2-pT602 or anti-Kv4.2-pT607 antibodies. Immunoprecipitation (IP) samples were blotted with Kv4.2 antibody. In WT mouse brains, pT607 Kv4.2 is almost half as abundant as pT602. However, PTZ administration increased the amount of pT607 until it reached a similar level as pT602. n = 4 for each group. T-test, **p < 0.01. f Mouse brain lysates from WT mice were incubated with excess anti-Kv4.2-pT602 or normal IgG antibodies. Immunoprecipitation (IP) samples were blotted with anti-Kv4.2 pT607 antibody. pT602 and pT607 dual phosphorylation was observed in mouse brain. Data was repeated in two independent experiments. g Mouse brain lysates from WT mice w or w/o PTZ administration (50 mg/kg, i.p., 15 min) were incubated with excess anti-Kv4.2-pT607 antibody. IP samples were blotted with anti-Kv4.2 pT602 antibody. PTZ-induced seizure increases the dual phosphorylation of T602 and T607 in mouse brain. Data was repeated in two independent experiments. Data are presented as mean ± SEM.

Fig 3: P38 phosphorylates Kv4.2 at a C-terminal Pin1 binding site.a P38 phosphorylates Kv4.2 at Thr602 and Thr607. Kv4.2 and p38a constructs (agf: p38 kinase dead mutant) were co-transfected into HEK-293T cells. Lysates were analyzed by western blotting with anti-pT602 and anti-pT607 specific antibodies. n = 15 for ctl, 9 for p38 and p38 agf. T-test, ***p < 0.001. b p38 binds to Kv4.2. Kv4.2 and p38a were co-transfected into HEK-293T cells. Detergent lysates were incubated with anti-Myc antibody and analyzed by western blotting with anti-Flag and anti-Myc antibodies. Data was repeated in two independent experiments. c, Enriched novel environment activates p38 in mouse hippocampus. n = 6 in each group. T-test, *p < 0.05. d KA-induced seizure activates p38 in mouse hippocampus. n = 8 in each group. T-test, ***p < 0.001. e PTZ-induced seizure activates p38 in mouse hippocampus. n = 6 for ctl and 5 for PTZ. T-test, ***p < 0.001. f SB203580, a potent p38 inhibitor (20 mg/kg, i.p., 15 min) blocked PTZ-induced phosphorylation of Kv4.2 T607 in mouse hippocampus. n = 4 in each group. T-test, **p < 0.01. g SL327, a selective MEK inhibitor (30 mg/kg, i.p., 15 min) did not block PTZ-induced phosphorylation of Kv4.2 T607 in mouse hippocampus. n = 7 in each group. *p < 0.05, **p < 0.01. t-test. Data are presented as mean ± SEM.

Fig 4: Phosphorylation of TRIM59 at Ser308 recruits PIN1. a IP and WB for TRIM59 binding to PIN1 in LN229/EGFR and U87/EGFR cells with or without EGF stimulation. b IP and WB for TRIM59 association with PIN1 in LN229/EGFR cells stimulated with EGF (100 ng/ml) for 6 h. Cells were pre-treated with or without Roscovitine (20 µM) for 1 h. c Effects of ectopic expression of TRIM59 WT, S308A, or S308D mutant on TRIM59 association with PIN1 with or without EGF stimulation. d Effects of ectopic expression of PIN1 WT or WW mutant on TRIM59 binding with PIN1. e Cis–trans isomerization analysis. The synthesized phosphorylated or nonphosphorylated oligopeptides of TRIM59 were mixed with purified PIN1 WT or C113A mutant. Data were expressed as means ± SD. P values were calculated using one-way analysis of variance (ANOVA). f WB for effect of PIN1 knockout with a PIN1 sgRNA on TRIM59 nuclear translocation in LN229/EGFR GBM cells. g Effects of re-expression of shRNA-resistant PIN1 WT or C113A mutant on TRIM59 nuclear translocation. LN229/EGFR GBM cells reconstituted with empty vector (EV), shRNA-resistant PIN1 WT, or C113A mutant were stimulated with or without EGF (100 ng/ml) for 6 h. Data are representative of three independent experiments with similar results. Source data are provided as a Source Data file

Fig 5: Pin1 influences Notch3 processing and stability in endogenous and exogenous system. (a) Western blot analysis of Notch3 extracellular (N3EC) and activated intracellular (N3IC-act) protein expression of whole-cell extract (WCE) derived from Pin1-silenced TALL-1 (+) vs control cells (-) (left panel). The western blots in the figure are representative of at least three independent experiments, each in triplicate. The optical densitometry (OD) (right panels) was analyzed in all the experiments performed, thus including the P-values, calculated using Student's T-test (i.e., **P?0.01). (b) WCEs from Pin1-silenced TALL-1 cells (+) vs control cells (-) in a time course assay with 10 µg/ml of cycloheximide (CHX), in the presence or absence of the proteasome inhibitor MG132 for the same times before lysis, were revealed by immunoblotting with anti-activated N3IC (N3IC-act), anti-Pin1 and anti-ß-actin antibodies (left panel). The right panel shows the relative quantification of activated-N3IC as determined by OD. (c) Left panel, Western blot analysis of whole-cell extracts from HEK293T cells transfected with Flag N3IC-wt plasmid and silenced for Pin1 (+) or control (-) in a time course assay with 10 µg/ml of cycloheximide (CHX). Extracts were immunoblotted with anti-Flag, anti-Pin1 and anti-ß-actin antibodies. The right panel shows the relative quantification of Flag N3IC as determined by OD. All data are representative of at least three independent experiments, each in triplicate.