1. Pin1 Mediates Neural-Specific Activation of the Mitochondrial Apoptotic Machinery 
Esther B.E. Becker, Azad Bonni 
Neuron 
Volume 49, Issue 5, Pages (March 2006)
DOI: /j.neuron
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1 Pin1 Interacts with Phosphorylated BIMEL In Vitro and In Vivo
(A) Lysates of 293T cells transfected with HA-BIMEL alone or together with FLAG-MEKK1Δ were subjected to a pulldown assay using GST or GST-Pin1, followed by immunoblotting with an anti-HA antibody. Lower panels show the expression of HA-BIMEL and FLAG-MEKK1Δ. (B) Lysates of 293T cells transfected with HA-BIMEL wild-type or HA-BIMEL S65A mutant with or without FLAG-MEKK1Δ were subjected to a GST-Pin1 pulldown assay as in (A). (C) Lysates of cerebellar granule neurons untreated or treated for 5 hr with 5 μM MG132 were subjected to a pulldown with GST-Pin1 WW, followed by immunoblotting for BIM. Lower panels show the expression of BIMEL and phosphorylated c-Jun. (D) Lysates from granule neurons that were kept in conditioned full medium (CM) or starved for 2 hr (Starv) were immunoprecipitated with a Pin1 antibody, followed by immunoblotting for Pin1, BIM, and Ser65-phosphorylated BIM. Nonspecific immunoreactivity with the IgG light chain is indicated by an asterisk (∗). Lower panel shows the expression of BIMEL.
Neuron  , DOI: ( /j.neuron )
Copyright © 2006 Elsevier Inc. Terms and Conditions

3. Figure 2
Pin1 Is Localized at the Mitochondria in Neurons and Associates with the Neuron-Specific JNK Signaling Scaffold Protein JIP3
(A) Nuclear (N), heavy membrane (HM), and cytosolic (C) fractions prepared from 293T cells, cortical neurons, and cerebellar granule neurons were subjected to immunoblotting for Pin1, BIM, the mitochondrial marker HSP60, and the nuclear marker SP1. (B) Cortical neurons were subjected to indirect immunofluorescence using antibodies to Pin1 and HSP60 and the DNA dye bisbenzimide (Hoechst 33258) and analyzed by confocal microscopy. (C) Whole-brain lysates were immunoprecipitated with a JIP3 antibody or control IgG, followed by immunoblotting for Pin1. Left panels show the expression of Pin1 and JIP3. (D) Lysates of 293T cells transfected with FLAG-tagged fragments of JIP3 fragment (1-442, , , and ) were subjected to a GST-Pin1 pulldown, followed by immunoblotting for FLAG and GST. Left panel shows the expression of FLAG-JIP3 fragments. (E) Lysates of granule neurons were subjected to a pulldown assay using GST or GST-Pin1 WW followed by immunoblotting for JIP3 and GST. (F) Lysates of granule neurons were incubated without or with λ phosphatase (PPase), followed by GST-Pin1 pulldown and immunoblotting for JIP3 and GST. (G) Lysates of 293T cells transfected with FLAG-JIP3 or FLAG-BIMEL together with FLAG-MEKK1Δ were subjected to a GST-Pin1 pulldown assay in the presence of increasing amount of Pintide peptide (0, 2.5, 5, 10, 25 mM), followed by immunoblotting for FLAG and GST. Representative immunoblots of three independent experiments are shown. (H) Lysates from granule neurons that were kept in conditioned full medium (CM) or starved for 2 hr (Starv) were immunoprecipitated using a JIP3 antibody, followed by immunoblotting for Pin1. Left panels show the expression of JIP3 and Pin1.
Neuron  , DOI: ( /j.neuron )
Copyright © 2006 Elsevier Inc. Terms and Conditions

4. Figure 3
Pin1 Contributes to Survival Factor Withdrawal-Induced and BIMEL-Induced Apoptosis in Neurons
(A) Granule neurons were transfected with the control vector or a Pin1 expression plasmid alone or together with a BCL-XL expression plasmid and a β-galactosidase expression vector. Neurons were fixed 40 hr after transfection and subjected to indirect immunofluorescence with a β-galactosidase antibody and the DNA dye bisbenzimide (Hoechst 33258). Percentage of apoptosis is represented as mean ± SEM. Expression of Pin1 significantly induced apoptosis (n = 3; ANOVA; p < 0.05), but was blocked by coexpression of BCL-XL (p < 0.01). (B) Lysates of 293T cells transfected with the control vector or Pin1 RNAi plasmid were subjected to immunoblotting for Pin1 and HSP60. (C) Granule neurons transfected with the Pin1 RNAi plasmid and β-galactosidase were subjected to indirect immunofluorescence using antibodies against β-galactosidase and Pin1 and the DNA dye bisbenzimide (Hoechst 33258). Arrowhead points at a transfected neuron with reduced Pin1 immunoreactivity. Endogenous Pin1 was robustly reduced in 50% of Pin1 RNAi-transfected neurons. (D) Granule neurons were transfected with the U6 control vector or U6/pin1 RNAi plasmid together with β-galactosidase. Four days after transfection, neurons were kept in conditioned full medium or starved for 24 hr and subjected to indirect immunofluorescence as in (A). Top panels show representative pictures of the starved neurons. Starvation significantly induced apoptosis in U6-transfected neurons (n = 5; ANOVA; p < ). Apoptosis upon starvation was significantly reduced in U6/pin1-transfected neurons (n = 5; ANOVA; p < ). (E) Granule neurons were transfected as in (D). Two days after transfection, neurons that were kept in full medium (BME + 5% serum + 25 mM KCl) or deprived of KCl for 48 hr were fixed and subjected to indirect immunofluorescence as in (A). KCl deprivation significantly induced apoptosis in U6-transfected neurons (n = 3; ANOVA; p < 0.005). Pin1 knockdown significantly protected neurons from KCl deprivation-induced apoptosis (n = 3; ANOVA; p < 0.005). (F) Granule neurons were transfected with the vector control, Pin1, or Pin1-Res plasmid together with the control U6 or U6/pin1 plasmid. Four days after transfection, neurons were starved for 16 hr and subjected to indirect immunofluorescence as in (A). Pin1 knockdown significantly reduced apoptosis (n = 3; ANOVA; p < 0.005). Expression of Pin1 failed to rescue the RNAi-induced phenotype, while expression of Pin1-Res significantly reversed the effect of Pin1 RNAi (n = 3; ANOVA; p < 0.001). (G) Granule neurons were transfected with the vector control or a BIMEL expression plasmid together with the control U6 or U6/pin1 RNAi plasmid. Four days after transfection, neurons were starved for 8 hr and subjected to indirect immunofluorescence as in (A). Expression of BIMEL significantly induced apoptosis (n = 3; ANOVA; p < ). BIMEL-induced apoptosis was significantly reduced upon Pin1 knockdown (n = 3; ANOVA; p < 0.005). (H) Granule neurons were transfected with the vector control or a BIMEL expression plasmid together with a Pin1 antisense plasmid. Neurons were treated as in (G). Expression of BIMEL significantly induced apoptosis (n = 3; ANOVA; p < ). Expression of Pin1 antisense RNA significantly reduced BIMEL-induced apoptosis (n = 3; ANOVA; p < 0.01).
Neuron  , DOI: ( /j.neuron )
Copyright © 2006 Elsevier Inc. Terms and Conditions

5. Figure 4
Pin1 Isomerase Activity Is Required for Neuronal Apoptosis and Stabilizes BIMEL Protein in Neurons
(A) Granule neurons were transfected with the vector control, a Pin1-Res, or a Pin1 WW S16A expression plasmid together with the control U6 or U6/pin1 plasmid and a β-galactosidase expression plasmid. Four days after transfection, neurons were starved for 16 hr and subjected to indirect immunofluorescence using antibodies against β-galactosidase and Pin1 and the DNA dye bisbenzimide (Hoechst 33258). Pin1 knockdown significantly reduced apoptosis (n = 6; ANOVA; p < ). Expression of Pin1-Res reversed the RNAi-induced phenotype (n = 6; ANOVA; p < ), while expression of Pin1 WW S16A had no significant effect. (B) Left panel: Lysates of 293T cells transfected with HA-BIMEL and FLAG-MEKK1Δ were subjected to a pulldown assay using GST, GST-Pin1, GST-Pin1 C113A, or GST-Pin1 H59A, followed by immunoblotting for HA or GST. Right panel: Granule neurons were transfected with the vector control, a Pin1-Res, a Pin1-Res C113A, or a Pin1-Res H59A expression plasmid together with the control U6 or U6/pin1 vector. Neurons were treated as in (A). Pin1 knockdown significantly reduced apoptosis (n = 3; ANOVA; p < ). Expression of Pin1-Res reversed the Pin1 RNAi-induced phenotype (n = 3; ANOVA; p < ), while expression of Pin1-Res C113A and H59A did not significantly affect the Pin1 RNAi-induced survival phenotype. (C) Granule neurons were infected with the vector control or Pin1 RNAi-expressing lentivirus. Four days after infection, neurons were starved for 2 hr, lysed, and lysates were subjected to immunoblotting for BIM, Pin1, GFP, and HSP60. Expression of the Pin1 RNAi plasmid significantly reduced Pin1 and BIMEL but not GFP or HSP60 protein levels (see text for details). (D) Granule neurons were infected with the vector control or Pin1 RNAi-expressing lentivirus. Four days after infection, RNA was isolated and subjected to RT-PCR with specific primers to BIMEL, Pin1, and GAPDH. (E) Model of Pin1's role in JNK-activation of BIMEL in neurons. See text for details.
Neuron  , DOI: ( /j.neuron )
Copyright © 2006 Elsevier Inc. Terms and Conditions