pin1 antibody / peptidyl-prolyl cis-trans isomerase nima-interacting 1 (NSJ Bioreagents)

NSJ Bioreagents pin1 antibody / peptidyl-prolyl cis-trans isomerase nima-interacting 1
Pin1 Antibody / Peptidyl Prolyl Cis Trans Isomerase Nima Interacting 1, supplied by NSJ Bioreagents, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/antibody+against+pin1/PIN1+Antibody+%2F+Peptidyl-prolyl+cis-trans+isomerase+NIMA-interacting+1/custom%40rq7631%4025951175
Average 99 stars, based on 1 article reviews

pin1 antibody / peptidyl-prolyl cis-trans isomerase nima-interacting 1 - by Bioz Stars, 2026-10

99/100 stars

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human/mouse pin1 antibody (Bio-Techne corporation)

Bio-Techne corporation human/mouse pin1 antibody
Human/Mouse Pin1 Antibody, supplied by Bio-Techne corporation, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/antibody+against+pin1/Human%2FMouse+Pin1+Antibody/bio-techne+corporation___mab2294
Average 93 stars, based on 1 article reviews

human/mouse pin1 antibody - by Bioz Stars, 2026-10

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anti pin1 (Santa Cruz Biotechnology)

Santa Cruz Biotechnology anti pin1
Anti Pin1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/antibody+against+pin1/Pin1+Antibody/pmc04815797-55-0-34
Average 94 stars, based on 1 article reviews

anti pin1 - by Bioz Stars, 2026-10

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antibody against pin1 (R&D Systems)

R&D Systems antibody against pin1

(a) Representative immunohistochemical staining of <t>Pin1</t> and RUNX3 in normal and tumor breast tissues. Boxed regions are enlarged below. The detection of the expression of RUNX3 and Pin1 of the human tissue microarray was performed as previously described . (b) Tissue sections of 80 breast cancer samples were immunostained with anti-Pin1 or anti-RUNX3 antibodies for the expression of Pin1 and RUNX3 and their correlation was analyzed by Spearman rank correlation test (p<0.01). (c) Expression levels of RUNX3 and Pin1 in various normal mammary and breast cancer cells. (d) RUNX3-MCF-7 cells stably expressing RUNX3 were transfected with control or Pin1 siRNA for 48 hr, whole cell lysates were immunoblotted as indicated for the expression levels of RUNX3 and Pin1. (d) MDA-MB-361 cells were transfected with control or Pin1 siRNA for 48 hr, the whole cell lysates were immunoblotted for the expression of endogenous RUNX3 and Pin1.

Antibody Against Pin1, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/antibody+against+pin1/Human%2FMouse+Pin1+Antibody/pmc03438320-194-0-5
Average 93 stars, based on 1 article reviews

antibody against pin1 - by Bioz Stars, 2026-10

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rabbit polyclonal antibody against pin1 (Proteintech)

Proteintech rabbit polyclonal antibody against pin1

FIGURE 1 The body weight (a) and blood glucose levels (b) of C57BL/6 mice were measured once a week. Blood samples were collected from the tails of mice under fasting conditions. Body weight was measured in grams, and blood glucose levels were measured using a blood glucose meter. The data are expressed as the mean ± SEM (n = 10). CON, normal control group; DB, diabetic group; DB + 0, sham treatment group; DB + <t>Pin1,</t> experimental group.

Rabbit Polyclonal Antibody Against Pin1, supplied by Proteintech, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/antibody+against+pin1/PIN1+Antibody/pm37990376-80-6-12
Average 94 stars, based on 1 article reviews

rabbit polyclonal antibody against pin1 - by Bioz Stars, 2026-10

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antibodies against phospho pin1 (Cell Signaling Technology Inc)

Cell Signaling Technology Inc antibodies against phospho pin1
$A) In vitro GST pull down assay. The cell lysates were incubated with GST or <t>GST-PIN1</t> fusion protein, followed by addition of the GST beads. The precipitates were immunoblotted with anti-HIF-1α to show the bound HIF-1α and reprobed with anti-GST to show the precipitated GST and GST-PIN1. B) Interaction of HIF-1α and PIN1 in HCT116 cells. Cells were transfected with Flag-tagged HIF-1α and HA-PIN1, and stimulated with hypoxia for 4 h. The cell lysates were immunoprecipitated with anti-Flag antibody, and the precipitates were fractionated by SDS-polyacrylamide gel electrophoresis and blotted with anti-HA antibody. C) Association of endogenous PIN1 and HIF-1α. HCT116 cells were subjected to hypoxia for 4 h. Cell lysates were incubated with either normal IgG or anti-PIN1 as labeled and blotted with anti HIF-1α. D) Binding of HIF-1α and PIN1 in situ . The HCT116 cells were incubated under normoxia or hypoxia. Interaction of HIF-1α and PIN1 was visualized by Duolink analysis. HIF-1α and PIN1 were co-labeled with antibodies. Nuclei were counter stained with DAPI (blue). Scale bar, 20 μm. E) ITC indicates that PiB binds PIN1. Heat evolution as a function of adding increasing amounts of PiB to GST-PIN1. The heats of dilution were measured separately, and found to be <1% of the signal at the start of titration. Fitting the ITC data with Microcal analysis launcher software indicates that PiB binds to GST-PIN1. Values shown on the figure are from the fit of the displayed dataset. K = 2.11E4 ± 4.46E3M -1 , N = 1.56 ± 0.0451 sites, ΔH = 3.977 ± 0.2676 Kcal/mol, ΔS = 33.1 cal/mol/deg. F) In vitro GST pull down assay. The cell lysates were incubated with GST or GST-PIN1 fusion protein, followed by treating with PiB and addition of the GST beads. The precipitates were immunoblotted with anti-HIF-1α to show the bound HIF-1α and reprobed with anti-GST to show the precipitated GST and GST-PIN1. G) Binding of HIF-1α and inactivated PIN1 in situ . HCT116 cells were incubated with or without PiB (20 μM). Interaction of HIF-1α and inactivated PIN1 was visualized by Duolink analysis. HIF-1α and PIN1 were co-labeled with antibodies. Nuclei were counter stained with DAPI (blue). H) CIP (50 U) was added to the supernatants at 30°C for the indicated time periods. Following incubation, GST or GST-PIN1 proteins were incubated with the supernatants for 4 h and then were pulled down with GST beads. Following incubation, reactions were stopped by the addition of SDS sample buffer, followed by SDS-PAGE. I) HA-HIF-1α proteins were purified using a commercially available kit. The proteins were treated with or without lamda phosphatase for 1 h at 30°C. Then GST-PIN1 proteins were incubated with purified HA-HIF-1α proteins and were pulled down with GST beads. The proteins were resolved in SDS-polyacrylamide gels and detected by immunoblotting. J) Cells were transfected with scrambled siRNA as a negative control or PIN1-siRNA for 72 h and treated with hypoxia for 4 h. Cell lysates were incubated with either normal IgG or anti-MPM2 as labeled and blotted with anti-PIN1. K) HA-HIF-1α, HA-HIF-1α S402/564A , and HA- HIF-1α S641/643A were transfected in HCT116 cells. Whole cell extracts of HCT116 cells were prepared for pull down assays with GST-PIN1 proteins. The pull downed fractions were s with anti-HA antibody.$
Antibodies Against Phospho Pin1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/antibody+against+pin1/Pin1+Antibody/pmc04718546-37-0-8
Average 95 stars, based on 1 article reviews

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c-myc antibody (NSJ Bioreagents)

NSJ Bioreagents c-myc antibody
$A) In vitro GST pull down assay. The cell lysates were incubated with GST or <t>GST-PIN1</t> fusion protein, followed by addition of the GST beads. The precipitates were immunoblotted with anti-HIF-1α to show the bound HIF-1α and reprobed with anti-GST to show the precipitated GST and GST-PIN1. B) Interaction of HIF-1α and PIN1 in HCT116 cells. Cells were transfected with Flag-tagged HIF-1α and HA-PIN1, and stimulated with hypoxia for 4 h. The cell lysates were immunoprecipitated with anti-Flag antibody, and the precipitates were fractionated by SDS-polyacrylamide gel electrophoresis and blotted with anti-HA antibody. C) Association of endogenous PIN1 and HIF-1α. HCT116 cells were subjected to hypoxia for 4 h. Cell lysates were incubated with either normal IgG or anti-PIN1 as labeled and blotted with anti HIF-1α. D) Binding of HIF-1α and PIN1 in situ . The HCT116 cells were incubated under normoxia or hypoxia. Interaction of HIF-1α and PIN1 was visualized by Duolink analysis. HIF-1α and PIN1 were co-labeled with antibodies. Nuclei were counter stained with DAPI (blue). Scale bar, 20 μm. E) ITC indicates that PiB binds PIN1. Heat evolution as a function of adding increasing amounts of PiB to GST-PIN1. The heats of dilution were measured separately, and found to be <1% of the signal at the start of titration. Fitting the ITC data with Microcal analysis launcher software indicates that PiB binds to GST-PIN1. Values shown on the figure are from the fit of the displayed dataset. K = 2.11E4 ± 4.46E3M -1 , N = 1.56 ± 0.0451 sites, ΔH = 3.977 ± 0.2676 Kcal/mol, ΔS = 33.1 cal/mol/deg. F) In vitro GST pull down assay. The cell lysates were incubated with GST or GST-PIN1 fusion protein, followed by treating with PiB and addition of the GST beads. The precipitates were immunoblotted with anti-HIF-1α to show the bound HIF-1α and reprobed with anti-GST to show the precipitated GST and GST-PIN1. G) Binding of HIF-1α and inactivated PIN1 in situ . HCT116 cells were incubated with or without PiB (20 μM). Interaction of HIF-1α and inactivated PIN1 was visualized by Duolink analysis. HIF-1α and PIN1 were co-labeled with antibodies. Nuclei were counter stained with DAPI (blue). H) CIP (50 U) was added to the supernatants at 30°C for the indicated time periods. Following incubation, GST or GST-PIN1 proteins were incubated with the supernatants for 4 h and then were pulled down with GST beads. Following incubation, reactions were stopped by the addition of SDS sample buffer, followed by SDS-PAGE. I) HA-HIF-1α proteins were purified using a commercially available kit. The proteins were treated with or without lamda phosphatase for 1 h at 30°C. Then GST-PIN1 proteins were incubated with purified HA-HIF-1α proteins and were pulled down with GST beads. The proteins were resolved in SDS-polyacrylamide gels and detected by immunoblotting. J) Cells were transfected with scrambled siRNA as a negative control or PIN1-siRNA for 72 h and treated with hypoxia for 4 h. Cell lysates were incubated with either normal IgG or anti-MPM2 as labeled and blotted with anti-PIN1. K) HA-HIF-1α, HA-HIF-1α S402/564A , and HA- HIF-1α S641/643A were transfected in HCT116 cells. Whole cell extracts of HCT116 cells were prepared for pull down assays with GST-PIN1 proteins. The pull downed fractions were s with anti-HA antibody.$
C Myc Antibody, supplied by NSJ Bioreagents, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/antibody+against+pin1/c-Myc+Antibody/custom%40f52056%4010%2E1074%2Fjbc%2Em804659200
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c-myc antibody - by Bioz Stars, 2026-10

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anti-cd74 antibody (StressMarq)

StressMarq anti-cd74 antibody
$A) In vitro GST pull down assay. The cell lysates were incubated with GST or <t>GST-PIN1</t> fusion protein, followed by addition of the GST beads. The precipitates were immunoblotted with anti-HIF-1α to show the bound HIF-1α and reprobed with anti-GST to show the precipitated GST and GST-PIN1. B) Interaction of HIF-1α and PIN1 in HCT116 cells. Cells were transfected with Flag-tagged HIF-1α and HA-PIN1, and stimulated with hypoxia for 4 h. The cell lysates were immunoprecipitated with anti-Flag antibody, and the precipitates were fractionated by SDS-polyacrylamide gel electrophoresis and blotted with anti-HA antibody. C) Association of endogenous PIN1 and HIF-1α. HCT116 cells were subjected to hypoxia for 4 h. Cell lysates were incubated with either normal IgG or anti-PIN1 as labeled and blotted with anti HIF-1α. D) Binding of HIF-1α and PIN1 in situ . The HCT116 cells were incubated under normoxia or hypoxia. Interaction of HIF-1α and PIN1 was visualized by Duolink analysis. HIF-1α and PIN1 were co-labeled with antibodies. Nuclei were counter stained with DAPI (blue). Scale bar, 20 μm. E) ITC indicates that PiB binds PIN1. Heat evolution as a function of adding increasing amounts of PiB to GST-PIN1. The heats of dilution were measured separately, and found to be <1% of the signal at the start of titration. Fitting the ITC data with Microcal analysis launcher software indicates that PiB binds to GST-PIN1. Values shown on the figure are from the fit of the displayed dataset. K = 2.11E4 ± 4.46E3M -1 , N = 1.56 ± 0.0451 sites, ΔH = 3.977 ± 0.2676 Kcal/mol, ΔS = 33.1 cal/mol/deg. F) In vitro GST pull down assay. The cell lysates were incubated with GST or GST-PIN1 fusion protein, followed by treating with PiB and addition of the GST beads. The precipitates were immunoblotted with anti-HIF-1α to show the bound HIF-1α and reprobed with anti-GST to show the precipitated GST and GST-PIN1. G) Binding of HIF-1α and inactivated PIN1 in situ . HCT116 cells were incubated with or without PiB (20 μM). Interaction of HIF-1α and inactivated PIN1 was visualized by Duolink analysis. HIF-1α and PIN1 were co-labeled with antibodies. Nuclei were counter stained with DAPI (blue). H) CIP (50 U) was added to the supernatants at 30°C for the indicated time periods. Following incubation, GST or GST-PIN1 proteins were incubated with the supernatants for 4 h and then were pulled down with GST beads. Following incubation, reactions were stopped by the addition of SDS sample buffer, followed by SDS-PAGE. I) HA-HIF-1α proteins were purified using a commercially available kit. The proteins were treated with or without lamda phosphatase for 1 h at 30°C. Then GST-PIN1 proteins were incubated with purified HA-HIF-1α proteins and were pulled down with GST beads. The proteins were resolved in SDS-polyacrylamide gels and detected by immunoblotting. J) Cells were transfected with scrambled siRNA as a negative control or PIN1-siRNA for 72 h and treated with hypoxia for 4 h. Cell lysates were incubated with either normal IgG or anti-MPM2 as labeled and blotted with anti-PIN1. K) HA-HIF-1α, HA-HIF-1α S402/564A , and HA- HIF-1α S641/643A were transfected in HCT116 cells. Whole cell extracts of HCT116 cells were prepared for pull down assays with GST-PIN1 proteins. The pull downed fractions were s with anti-HA antibody.$
Anti Cd74 Antibody, supplied by StressMarq, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/antibody+against+pin1/Anti-CD74+Antibody/stressmarq___smc-116
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plk1 antibody (NSJ Bioreagents)

NSJ Bioreagents plk1 antibody
$A) In vitro GST pull down assay. The cell lysates were incubated with GST or <t>GST-PIN1</t> fusion protein, followed by addition of the GST beads. The precipitates were immunoblotted with anti-HIF-1α to show the bound HIF-1α and reprobed with anti-GST to show the precipitated GST and GST-PIN1. B) Interaction of HIF-1α and PIN1 in HCT116 cells. Cells were transfected with Flag-tagged HIF-1α and HA-PIN1, and stimulated with hypoxia for 4 h. The cell lysates were immunoprecipitated with anti-Flag antibody, and the precipitates were fractionated by SDS-polyacrylamide gel electrophoresis and blotted with anti-HA antibody. C) Association of endogenous PIN1 and HIF-1α. HCT116 cells were subjected to hypoxia for 4 h. Cell lysates were incubated with either normal IgG or anti-PIN1 as labeled and blotted with anti HIF-1α. D) Binding of HIF-1α and PIN1 in situ . The HCT116 cells were incubated under normoxia or hypoxia. Interaction of HIF-1α and PIN1 was visualized by Duolink analysis. HIF-1α and PIN1 were co-labeled with antibodies. Nuclei were counter stained with DAPI (blue). Scale bar, 20 μm. E) ITC indicates that PiB binds PIN1. Heat evolution as a function of adding increasing amounts of PiB to GST-PIN1. The heats of dilution were measured separately, and found to be <1% of the signal at the start of titration. Fitting the ITC data with Microcal analysis launcher software indicates that PiB binds to GST-PIN1. Values shown on the figure are from the fit of the displayed dataset. K = 2.11E4 ± 4.46E3M -1 , N = 1.56 ± 0.0451 sites, ΔH = 3.977 ± 0.2676 Kcal/mol, ΔS = 33.1 cal/mol/deg. F) In vitro GST pull down assay. The cell lysates were incubated with GST or GST-PIN1 fusion protein, followed by treating with PiB and addition of the GST beads. The precipitates were immunoblotted with anti-HIF-1α to show the bound HIF-1α and reprobed with anti-GST to show the precipitated GST and GST-PIN1. G) Binding of HIF-1α and inactivated PIN1 in situ . HCT116 cells were incubated with or without PiB (20 μM). Interaction of HIF-1α and inactivated PIN1 was visualized by Duolink analysis. HIF-1α and PIN1 were co-labeled with antibodies. Nuclei were counter stained with DAPI (blue). H) CIP (50 U) was added to the supernatants at 30°C for the indicated time periods. Following incubation, GST or GST-PIN1 proteins were incubated with the supernatants for 4 h and then were pulled down with GST beads. Following incubation, reactions were stopped by the addition of SDS sample buffer, followed by SDS-PAGE. I) HA-HIF-1α proteins were purified using a commercially available kit. The proteins were treated with or without lamda phosphatase for 1 h at 30°C. Then GST-PIN1 proteins were incubated with purified HA-HIF-1α proteins and were pulled down with GST beads. The proteins were resolved in SDS-polyacrylamide gels and detected by immunoblotting. J) Cells were transfected with scrambled siRNA as a negative control or PIN1-siRNA for 72 h and treated with hypoxia for 4 h. Cell lysates were incubated with either normal IgG or anti-MPM2 as labeled and blotted with anti-PIN1. K) HA-HIF-1α, HA-HIF-1α S402/564A , and HA- HIF-1α S641/643A were transfected in HCT116 cells. Whole cell extracts of HCT116 cells were prepared for pull down assays with GST-PIN1 proteins. The pull downed fractions were s with anti-HA antibody.$
Plk1 Antibody, supplied by NSJ Bioreagents, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/antibody+against+pin1/PLK1+Antibody/custom%40rq5512%4029676205
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antibodies against mitotic phosphoproteins (US Biological Life Sciences)

US Biological Life Sciences antibodies against mitotic phosphoproteins
$A) In vitro GST pull down assay. The cell lysates were incubated with GST or <t>GST-PIN1</t> fusion protein, followed by addition of the GST beads. The precipitates were immunoblotted with anti-HIF-1α to show the bound HIF-1α and reprobed with anti-GST to show the precipitated GST and GST-PIN1. B) Interaction of HIF-1α and PIN1 in HCT116 cells. Cells were transfected with Flag-tagged HIF-1α and HA-PIN1, and stimulated with hypoxia for 4 h. The cell lysates were immunoprecipitated with anti-Flag antibody, and the precipitates were fractionated by SDS-polyacrylamide gel electrophoresis and blotted with anti-HA antibody. C) Association of endogenous PIN1 and HIF-1α. HCT116 cells were subjected to hypoxia for 4 h. Cell lysates were incubated with either normal IgG or anti-PIN1 as labeled and blotted with anti HIF-1α. D) Binding of HIF-1α and PIN1 in situ . The HCT116 cells were incubated under normoxia or hypoxia. Interaction of HIF-1α and PIN1 was visualized by Duolink analysis. HIF-1α and PIN1 were co-labeled with antibodies. Nuclei were counter stained with DAPI (blue). Scale bar, 20 μm. E) ITC indicates that PiB binds PIN1. Heat evolution as a function of adding increasing amounts of PiB to GST-PIN1. The heats of dilution were measured separately, and found to be <1% of the signal at the start of titration. Fitting the ITC data with Microcal analysis launcher software indicates that PiB binds to GST-PIN1. Values shown on the figure are from the fit of the displayed dataset. K = 2.11E4 ± 4.46E3M -1 , N = 1.56 ± 0.0451 sites, ΔH = 3.977 ± 0.2676 Kcal/mol, ΔS = 33.1 cal/mol/deg. F) In vitro GST pull down assay. The cell lysates were incubated with GST or GST-PIN1 fusion protein, followed by treating with PiB and addition of the GST beads. The precipitates were immunoblotted with anti-HIF-1α to show the bound HIF-1α and reprobed with anti-GST to show the precipitated GST and GST-PIN1. G) Binding of HIF-1α and inactivated PIN1 in situ . HCT116 cells were incubated with or without PiB (20 μM). Interaction of HIF-1α and inactivated PIN1 was visualized by Duolink analysis. HIF-1α and PIN1 were co-labeled with antibodies. Nuclei were counter stained with DAPI (blue). H) CIP (50 U) was added to the supernatants at 30°C for the indicated time periods. Following incubation, GST or GST-PIN1 proteins were incubated with the supernatants for 4 h and then were pulled down with GST beads. Following incubation, reactions were stopped by the addition of SDS sample buffer, followed by SDS-PAGE. I) HA-HIF-1α proteins were purified using a commercially available kit. The proteins were treated with or without lamda phosphatase for 1 h at 30°C. Then GST-PIN1 proteins were incubated with purified HA-HIF-1α proteins and were pulled down with GST beads. The proteins were resolved in SDS-polyacrylamide gels and detected by immunoblotting. J) Cells were transfected with scrambled siRNA as a negative control or PIN1-siRNA for 72 h and treated with hypoxia for 4 h. Cell lysates were incubated with either normal IgG or anti-MPM2 as labeled and blotted with anti-PIN1. K) HA-HIF-1α, HA-HIF-1α S402/564A , and HA- HIF-1α S641/643A were transfected in HCT116 cells. Whole cell extracts of HCT116 cells were prepared for pull down assays with GST-PIN1 proteins. The pull downed fractions were s with anti-HA antibody.$
Antibodies Against Mitotic Phosphoproteins, supplied by US Biological Life Sciences, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/antibody+against+pin1/antibodies+against+mitotic+phosphoproteins/pm17466629-254-22-25
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anti mouse (Bethyl)

Bethyl anti mouse
$A) In vitro GST pull down assay. The cell lysates were incubated with GST or <t>GST-PIN1</t> fusion protein, followed by addition of the GST beads. The precipitates were immunoblotted with anti-HIF-1α to show the bound HIF-1α and reprobed with anti-GST to show the precipitated GST and GST-PIN1. B) Interaction of HIF-1α and PIN1 in HCT116 cells. Cells were transfected with Flag-tagged HIF-1α and HA-PIN1, and stimulated with hypoxia for 4 h. The cell lysates were immunoprecipitated with anti-Flag antibody, and the precipitates were fractionated by SDS-polyacrylamide gel electrophoresis and blotted with anti-HA antibody. C) Association of endogenous PIN1 and HIF-1α. HCT116 cells were subjected to hypoxia for 4 h. Cell lysates were incubated with either normal IgG or anti-PIN1 as labeled and blotted with anti HIF-1α. D) Binding of HIF-1α and PIN1 in situ . The HCT116 cells were incubated under normoxia or hypoxia. Interaction of HIF-1α and PIN1 was visualized by Duolink analysis. HIF-1α and PIN1 were co-labeled with antibodies. Nuclei were counter stained with DAPI (blue). Scale bar, 20 μm. E) ITC indicates that PiB binds PIN1. Heat evolution as a function of adding increasing amounts of PiB to GST-PIN1. The heats of dilution were measured separately, and found to be <1% of the signal at the start of titration. Fitting the ITC data with Microcal analysis launcher software indicates that PiB binds to GST-PIN1. Values shown on the figure are from the fit of the displayed dataset. K = 2.11E4 ± 4.46E3M -1 , N = 1.56 ± 0.0451 sites, ΔH = 3.977 ± 0.2676 Kcal/mol, ΔS = 33.1 cal/mol/deg. F) In vitro GST pull down assay. The cell lysates were incubated with GST or GST-PIN1 fusion protein, followed by treating with PiB and addition of the GST beads. The precipitates were immunoblotted with anti-HIF-1α to show the bound HIF-1α and reprobed with anti-GST to show the precipitated GST and GST-PIN1. G) Binding of HIF-1α and inactivated PIN1 in situ . HCT116 cells were incubated with or without PiB (20 μM). Interaction of HIF-1α and inactivated PIN1 was visualized by Duolink analysis. HIF-1α and PIN1 were co-labeled with antibodies. Nuclei were counter stained with DAPI (blue). H) CIP (50 U) was added to the supernatants at 30°C for the indicated time periods. Following incubation, GST or GST-PIN1 proteins were incubated with the supernatants for 4 h and then were pulled down with GST beads. Following incubation, reactions were stopped by the addition of SDS sample buffer, followed by SDS-PAGE. I) HA-HIF-1α proteins were purified using a commercially available kit. The proteins were treated with or without lamda phosphatase for 1 h at 30°C. Then GST-PIN1 proteins were incubated with purified HA-HIF-1α proteins and were pulled down with GST beads. The proteins were resolved in SDS-polyacrylamide gels and detected by immunoblotting. J) Cells were transfected with scrambled siRNA as a negative control or PIN1-siRNA for 72 h and treated with hypoxia for 4 h. Cell lysates were incubated with either normal IgG or anti-MPM2 as labeled and blotted with anti-PIN1. K) HA-HIF-1α, HA-HIF-1α S402/564A , and HA- HIF-1α S641/643A were transfected in HCT116 cells. Whole cell extracts of HCT116 cells were prepared for pull down assays with GST-PIN1 proteins. The pull downed fractions were s with anti-HA antibody.$
Anti Mouse, supplied by Bethyl, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/antibody+against+pin1/Mouse+Albumin+Antibody/10__1128_slash_jvi__02877___13-94-57-58
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anti mouse - by Bioz Stars, 2026-10

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ha tag antibody (NSJ Bioreagents)

NSJ Bioreagents ha tag antibody
$A) In vitro GST pull down assay. The cell lysates were incubated with GST or <t>GST-PIN1</t> fusion protein, followed by addition of the GST beads. The precipitates were immunoblotted with anti-HIF-1α to show the bound HIF-1α and reprobed with anti-GST to show the precipitated GST and GST-PIN1. B) Interaction of HIF-1α and PIN1 in HCT116 cells. Cells were transfected with Flag-tagged HIF-1α and HA-PIN1, and stimulated with hypoxia for 4 h. The cell lysates were immunoprecipitated with anti-Flag antibody, and the precipitates were fractionated by SDS-polyacrylamide gel electrophoresis and blotted with anti-HA antibody. C) Association of endogenous PIN1 and HIF-1α. HCT116 cells were subjected to hypoxia for 4 h. Cell lysates were incubated with either normal IgG or anti-PIN1 as labeled and blotted with anti HIF-1α. D) Binding of HIF-1α and PIN1 in situ . The HCT116 cells were incubated under normoxia or hypoxia. Interaction of HIF-1α and PIN1 was visualized by Duolink analysis. HIF-1α and PIN1 were co-labeled with antibodies. Nuclei were counter stained with DAPI (blue). Scale bar, 20 μm. E) ITC indicates that PiB binds PIN1. Heat evolution as a function of adding increasing amounts of PiB to GST-PIN1. The heats of dilution were measured separately, and found to be <1% of the signal at the start of titration. Fitting the ITC data with Microcal analysis launcher software indicates that PiB binds to GST-PIN1. Values shown on the figure are from the fit of the displayed dataset. K = 2.11E4 ± 4.46E3M -1 , N = 1.56 ± 0.0451 sites, ΔH = 3.977 ± 0.2676 Kcal/mol, ΔS = 33.1 cal/mol/deg. F) In vitro GST pull down assay. The cell lysates were incubated with GST or GST-PIN1 fusion protein, followed by treating with PiB and addition of the GST beads. The precipitates were immunoblotted with anti-HIF-1α to show the bound HIF-1α and reprobed with anti-GST to show the precipitated GST and GST-PIN1. G) Binding of HIF-1α and inactivated PIN1 in situ . HCT116 cells were incubated with or without PiB (20 μM). Interaction of HIF-1α and inactivated PIN1 was visualized by Duolink analysis. HIF-1α and PIN1 were co-labeled with antibodies. Nuclei were counter stained with DAPI (blue). H) CIP (50 U) was added to the supernatants at 30°C for the indicated time periods. Following incubation, GST or GST-PIN1 proteins were incubated with the supernatants for 4 h and then were pulled down with GST beads. Following incubation, reactions were stopped by the addition of SDS sample buffer, followed by SDS-PAGE. I) HA-HIF-1α proteins were purified using a commercially available kit. The proteins were treated with or without lamda phosphatase for 1 h at 30°C. Then GST-PIN1 proteins were incubated with purified HA-HIF-1α proteins and were pulled down with GST beads. The proteins were resolved in SDS-polyacrylamide gels and detected by immunoblotting. J) Cells were transfected with scrambled siRNA as a negative control or PIN1-siRNA for 72 h and treated with hypoxia for 4 h. Cell lysates were incubated with either normal IgG or anti-MPM2 as labeled and blotted with anti-PIN1. K) HA-HIF-1α, HA-HIF-1α S402/564A , and HA- HIF-1α S641/643A were transfected in HCT116 cells. Whole cell extracts of HCT116 cells were prepared for pull down assays with GST-PIN1 proteins. The pull downed fractions were s with anti-HA antibody.$
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(a) Representative immunohistochemical staining of Pin1 and RUNX3 in normal and tumor breast tissues. Boxed regions are enlarged below. The detection of the expression of RUNX3 and Pin1 of the human tissue microarray was performed as previously described . (b) Tissue sections of 80 breast cancer samples were immunostained with anti-Pin1 or anti-RUNX3 antibodies for the expression of Pin1 and RUNX3 and their correlation was analyzed by Spearman rank correlation test (p<0.01). (c) Expression levels of RUNX3 and Pin1 in various normal mammary and breast cancer cells. (d) RUNX3-MCF-7 cells stably expressing RUNX3 were transfected with control or Pin1 siRNA for 48 hr, whole cell lysates were immunoblotted as indicated for the expression levels of RUNX3 and Pin1. (d) MDA-MB-361 cells were transfected with control or Pin1 siRNA for 48 hr, the whole cell lysates were immunoblotted for the expression of endogenous RUNX3 and Pin1.

Journal: Oncogene

Article Title: Prolyl isomerase Pin1 down-regulates tumor suppressor RUNX3 in breast cancer

doi: 10.1038/onc.2012.178

Figure Lengend Snippet: (a) Representative immunohistochemical staining of Pin1 and RUNX3 in normal and tumor breast tissues. Boxed regions are enlarged below. The detection of the expression of RUNX3 and Pin1 of the human tissue microarray was performed as previously described . (b) Tissue sections of 80 breast cancer samples were immunostained with anti-Pin1 or anti-RUNX3 antibodies for the expression of Pin1 and RUNX3 and their correlation was analyzed by Spearman rank correlation test (p<0.01). (c) Expression levels of RUNX3 and Pin1 in various normal mammary and breast cancer cells. (d) RUNX3-MCF-7 cells stably expressing RUNX3 were transfected with control or Pin1 siRNA for 48 hr, whole cell lysates were immunoblotted as indicated for the expression levels of RUNX3 and Pin1. (d) MDA-MB-361 cells were transfected with control or Pin1 siRNA for 48 hr, the whole cell lysates were immunoblotted for the expression of endogenous RUNX3 and Pin1.

Article Snippet: Antibody against Pin1 was from R&D systems (Minneapolis, MN, USA), and antibody against RUNX3 was from Abcam (Cambridge, MA, USA).

Techniques: Immunohistochemical staining, Staining, Expressing, Microarray, Stable Transfection, Transfection, Control

(a) Pin1 interacts with RUNX3 in vivo. HEK293T cells were transfected with Flag-tagged Pin1 and Myc-tagged RUNX3 as indicated. 40 hr post-transfection, Flag-Pin1 immunoprecipitates were prepared from whole-cell lysates and immunoblotted for Myc-RUNX3. (b) Endogenous Pin1 interacts with endogenous RUNX3. Endogenous Pin1 immunoprecipitates were prepared from whole-cell lysates of MDA-MB-157 cells and immunoblotted for associated RUNX3. IgG was used as a control. (c) Pin1 interacts with RUNX3 in vitro. GST or GTS-Pin1 conjugated agaroses were incubated with HEK293T cell lysates transfected with Flag-RUNX3. The recovered materials were immunoblotted for Flag-RUNX3. (d) Pin1 interacts with phosphorylated RUNX3 in vitro. HEK293T cell lysates containing Flag-RUNX3 were pretreated with calf intestinal phosphatase (CIP) for 30 min followed by the incubation with GST-Pin1 conjugated agarose. GST-Pin1 associated RUNX3 was detected as in (c). Phosphorylated RUNX3 was immunoblotted with anti-pSer/Thr-Pro (MPM2) antibody. (e) RUNX3 contains pSer/Thr-Pro motif. Cell lysates from RUNX3-7 cells were immunoprecipitated with IgG or anti-MPM2 antibody and immunoblotted for the associated RUNX3 proteins.

Journal: Oncogene

Article Title: Prolyl isomerase Pin1 down-regulates tumor suppressor RUNX3 in breast cancer

doi: 10.1038/onc.2012.178

Figure Lengend Snippet: (a) Pin1 interacts with RUNX3 in vivo. HEK293T cells were transfected with Flag-tagged Pin1 and Myc-tagged RUNX3 as indicated. 40 hr post-transfection, Flag-Pin1 immunoprecipitates were prepared from whole-cell lysates and immunoblotted for Myc-RUNX3. (b) Endogenous Pin1 interacts with endogenous RUNX3. Endogenous Pin1 immunoprecipitates were prepared from whole-cell lysates of MDA-MB-157 cells and immunoblotted for associated RUNX3. IgG was used as a control. (c) Pin1 interacts with RUNX3 in vitro. GST or GTS-Pin1 conjugated agaroses were incubated with HEK293T cell lysates transfected with Flag-RUNX3. The recovered materials were immunoblotted for Flag-RUNX3. (d) Pin1 interacts with phosphorylated RUNX3 in vitro. HEK293T cell lysates containing Flag-RUNX3 were pretreated with calf intestinal phosphatase (CIP) for 30 min followed by the incubation with GST-Pin1 conjugated agarose. GST-Pin1 associated RUNX3 was detected as in (c). Phosphorylated RUNX3 was immunoblotted with anti-pSer/Thr-Pro (MPM2) antibody. (e) RUNX3 contains pSer/Thr-Pro motif. Cell lysates from RUNX3-7 cells were immunoprecipitated with IgG or anti-MPM2 antibody and immunoblotted for the associated RUNX3 proteins.

Article Snippet: Antibody against Pin1 was from R&D systems (Minneapolis, MN, USA), and antibody against RUNX3 was from Abcam (Cambridge, MA, USA).

Techniques: In Vivo, Transfection, Control, In Vitro, Incubation, Immunoprecipitation

(a) WW domain of Pin1 is involved in its association with RUNX3. HEK293T cell lysates containing transfected Flag-RUNX3 were subjected to GST pull-down assay with wild-type Pin1 and various Pin1 mutants. (b) HEK293T cells were co-transfected with expression vectors for Myc-Pin1 or Myc-Pin1-W34A and Flag-RUNX3 as indicated. Flag-RUNX3 immunoprecipitates were prepared from whole-cell lysates and immunoblotted for Myc-Pin1 and its mutant. (c) The region of RUNX3 from amino acids 188 to 234 is important for its interaction with Pin1. Left: Schematic showing the domain structure of RUNX3 and various deletion mutants of RUNX3. Right: GST-Pin1 conjugated agarose was incubated with cell lysates containing transfected Flag-RUNX3 or its various deletion mutants as indicated. The recovered materials were immunoblotted for RUNX3 and its deletion mutants with anti-Flag antibodies. Levels of GST or GST-Pin1 were shown by coomassie brilliant blue staining (CBB). (d) Four Ser/Thr-Pro motifs in RUNX3 are important for its interaction with Pin1. GST-Pin1 conjugated agarose was incubated with cell lysates containing transfected Flag-RUNX3 or its various point mutation mutants as indicated. The recovered materials were immunoblotted for RUNX3 and its mutants with anti-Flag antibodies. (e) HEK293T cells were transfected with expression vectors for Myc-Pin1 and Flag-RUNX3 or Flag-RUNX3-4A as indicated. 40 hr post-transfection, Flag-RUNX3 immunoprecipitates were prepared from whole-cell lysates and immunoblotted for Pin1 with anti-Myc antibodies.

Journal: Oncogene

Article Title: Prolyl isomerase Pin1 down-regulates tumor suppressor RUNX3 in breast cancer

doi: 10.1038/onc.2012.178

Figure Lengend Snippet: (a) WW domain of Pin1 is involved in its association with RUNX3. HEK293T cell lysates containing transfected Flag-RUNX3 were subjected to GST pull-down assay with wild-type Pin1 and various Pin1 mutants. (b) HEK293T cells were co-transfected with expression vectors for Myc-Pin1 or Myc-Pin1-W34A and Flag-RUNX3 as indicated. Flag-RUNX3 immunoprecipitates were prepared from whole-cell lysates and immunoblotted for Myc-Pin1 and its mutant. (c) The region of RUNX3 from amino acids 188 to 234 is important for its interaction with Pin1. Left: Schematic showing the domain structure of RUNX3 and various deletion mutants of RUNX3. Right: GST-Pin1 conjugated agarose was incubated with cell lysates containing transfected Flag-RUNX3 or its various deletion mutants as indicated. The recovered materials were immunoblotted for RUNX3 and its deletion mutants with anti-Flag antibodies. Levels of GST or GST-Pin1 were shown by coomassie brilliant blue staining (CBB). (d) Four Ser/Thr-Pro motifs in RUNX3 are important for its interaction with Pin1. GST-Pin1 conjugated agarose was incubated with cell lysates containing transfected Flag-RUNX3 or its various point mutation mutants as indicated. The recovered materials were immunoblotted for RUNX3 and its mutants with anti-Flag antibodies. (e) HEK293T cells were transfected with expression vectors for Myc-Pin1 and Flag-RUNX3 or Flag-RUNX3-4A as indicated. 40 hr post-transfection, Flag-RUNX3 immunoprecipitates were prepared from whole-cell lysates and immunoblotted for Pin1 with anti-Myc antibodies.

Article Snippet: Antibody against Pin1 was from R&D systems (Minneapolis, MN, USA), and antibody against RUNX3 was from Abcam (Cambridge, MA, USA).

Techniques: Transfection, Pull Down Assay, Expressing, Mutagenesis, Incubation, Staining

(a) Pin1 inhibits the transcriptional activity of RUNX3 in a prolyl isomerase activity-dependent manner. TβRE reporter plasmids were cotransfected with TβRI(CA) and RUNX3 expression vectors, alone or in combination with increasing amounts of Pin1 and its mutants into HEK293T cells. Luciferase activity was measured 30 hr after transfection. In each experiment, cells were also co-transfected with Renilla luciferase reporter plasmid which was used as an internal control. Results represent the average of three independent experiments +/− SD. Statistical analysis (p values) was performed using the Analysis of Variance (ANOVA) test. (b) Pin1 inhibits the transcriptional activity of RUNX3 in MCF-7 cells. MCF-7 cells were transfected, luciferase assay and statistical analysis were performed as in (a). (c) Pin1 inhibits the transcriptional activity of RUNX3 but not RUNX3-4A. TβRE reporter plasmids were co-transfected with TβRI(CA) and expression vectors for RUNX3 or RUNX3-4A, alone or in combination with Pin1 into HEK293T cells. Luciferase assay was performed as in (a). Statistical analysis was performed using a student’s t-test for 2 variables. (d) Depletion of Pin1 enhances transcriptional activity of RUNX3. MCF-7 cells were transfected with control or Pin1 siRNA. 48 hr after transfection, TβRE reporter plasmids were co-transfected with TβRI(CA) and expression vectors for RUNX3. Luciferase assay and statistical analysis were performed as in (a).

Journal: Oncogene

Article Title: Prolyl isomerase Pin1 down-regulates tumor suppressor RUNX3 in breast cancer

doi: 10.1038/onc.2012.178

Figure Lengend Snippet: (a) Pin1 inhibits the transcriptional activity of RUNX3 in a prolyl isomerase activity-dependent manner. TβRE reporter plasmids were cotransfected with TβRI(CA) and RUNX3 expression vectors, alone or in combination with increasing amounts of Pin1 and its mutants into HEK293T cells. Luciferase activity was measured 30 hr after transfection. In each experiment, cells were also co-transfected with Renilla luciferase reporter plasmid which was used as an internal control. Results represent the average of three independent experiments +/− SD. Statistical analysis (p values) was performed using the Analysis of Variance (ANOVA) test. (b) Pin1 inhibits the transcriptional activity of RUNX3 in MCF-7 cells. MCF-7 cells were transfected, luciferase assay and statistical analysis were performed as in (a). (c) Pin1 inhibits the transcriptional activity of RUNX3 but not RUNX3-4A. TβRE reporter plasmids were co-transfected with TβRI(CA) and expression vectors for RUNX3 or RUNX3-4A, alone or in combination with Pin1 into HEK293T cells. Luciferase assay was performed as in (a). Statistical analysis was performed using a student’s t-test for 2 variables. (d) Depletion of Pin1 enhances transcriptional activity of RUNX3. MCF-7 cells were transfected with control or Pin1 siRNA. 48 hr after transfection, TβRE reporter plasmids were co-transfected with TβRI(CA) and expression vectors for RUNX3. Luciferase assay and statistical analysis were performed as in (a).

Article Snippet: Antibody against Pin1 was from R&D systems (Minneapolis, MN, USA), and antibody against RUNX3 was from Abcam (Cambridge, MA, USA).

Techniques: Activity Assay, Expressing, Luciferase, Transfection, Plasmid Preparation, Control

(a) Pin1 reduces the cellular levels of RUNX3. RUNX3-MCF-7 cells were transiently transfected with expression vectors for Flag-Pin1 or Flag-Pin1-C115A. Levels of RUNX3 were detected by immunoblotting the whole-cell extracts with anti-Flag antibodies. Levels of Pin1 and tubulin were shown as indicated. (b) Pin1 induces proteasome-mediated degradation of RUNX3. RUNX3-MCF-7 cells were transfected with Flag-Pin1 as in (a). 24 hr after transfection, cells were treated with increasing doses of MG132 for 8 hr. Cell lysates were then immunoblotted for expression of RUNX3, Pin1 and tubulin. (c) Pin1 does not alter the mRNA levels of RUNX3. RUNX3-MCF-7 cells were transfected with Flag-Pin1 for 48 hr before total RNA was extracted. Complementary DNA was synthesized and quantitative real-time PCR was performed. Levels of RUNX3 mRNA were normalized with the expression of hGAPDH. Statistical analysis was performed using a student’s t-test. (d) Interaction between Pin1 and RUNX3 is critical for the degradation of RUNX3. MCF-7 cells were transiently transfected with expression vectors for Flag-Pin1, Flag-RUNX3 or Flag-RUNX3-4A. Cell lysates were immunoblotted for RUNX3 and Pin1 as in (a). (e) Pin1 reduces the half-life of RUNX3. HEK293T cells were transfected with wild-type RUNX3 or RUNX3-4A with or without Pin1 or Pin1-C115A as indicated. 24 hr after transfection, cells were treated with 100 ng/ml of cycloheximide (CHX) for the indicated time points, and immunoblotted for the expression of Flag-RUNX3. A representative result from three independent experiments is shown in the left panels. Quantification of the results is shown in the right panel. Data represent the average of three independent experiments +/− SD.

Journal: Oncogene

Article Title: Prolyl isomerase Pin1 down-regulates tumor suppressor RUNX3 in breast cancer

doi: 10.1038/onc.2012.178

Figure Lengend Snippet: (a) Pin1 reduces the cellular levels of RUNX3. RUNX3-MCF-7 cells were transiently transfected with expression vectors for Flag-Pin1 or Flag-Pin1-C115A. Levels of RUNX3 were detected by immunoblotting the whole-cell extracts with anti-Flag antibodies. Levels of Pin1 and tubulin were shown as indicated. (b) Pin1 induces proteasome-mediated degradation of RUNX3. RUNX3-MCF-7 cells were transfected with Flag-Pin1 as in (a). 24 hr after transfection, cells were treated with increasing doses of MG132 for 8 hr. Cell lysates were then immunoblotted for expression of RUNX3, Pin1 and tubulin. (c) Pin1 does not alter the mRNA levels of RUNX3. RUNX3-MCF-7 cells were transfected with Flag-Pin1 for 48 hr before total RNA was extracted. Complementary DNA was synthesized and quantitative real-time PCR was performed. Levels of RUNX3 mRNA were normalized with the expression of hGAPDH. Statistical analysis was performed using a student’s t-test. (d) Interaction between Pin1 and RUNX3 is critical for the degradation of RUNX3. MCF-7 cells were transiently transfected with expression vectors for Flag-Pin1, Flag-RUNX3 or Flag-RUNX3-4A. Cell lysates were immunoblotted for RUNX3 and Pin1 as in (a). (e) Pin1 reduces the half-life of RUNX3. HEK293T cells were transfected with wild-type RUNX3 or RUNX3-4A with or without Pin1 or Pin1-C115A as indicated. 24 hr after transfection, cells were treated with 100 ng/ml of cycloheximide (CHX) for the indicated time points, and immunoblotted for the expression of Flag-RUNX3. A representative result from three independent experiments is shown in the left panels. Quantification of the results is shown in the right panel. Data represent the average of three independent experiments +/− SD.

Article Snippet: Antibody against Pin1 was from R&D systems (Minneapolis, MN, USA), and antibody against RUNX3 was from Abcam (Cambridge, MA, USA).

Techniques: Transfection, Expressing, Western Blot, Synthesized, Real-time Polymerase Chain Reaction

(a) Pin1 enhances ubiquitination of RUNX3. HEK293T cells were transfected with indicated combinations of plasmids expressing Flag-RUNX3, HA-ubiquitin, Myc-Pin1 or Myc-Pin1-C115A. RUNX3 immunoprecipitates were immunoblotted for ubiquitination with anti-HA antibodies. Levels of RUNX3 and Pin1 are shown in the lower two panels. (b) Interaction between Pin1 and RUNX3 is important for the Pin1-induced ubiquitination of RUNX3. HEK293T cells were transfected with a combination of Flag-RUNX3 or Flag-RUNX3-4A, HA-ubiquitin and Myc-Pin1 as indicated. RUNX3 ubiquitination was detected as in (a). (c) Down-regulation of Pin1 decreases the ubiquitination of RUNX3. RUNX3-MCF-7 cells were transfected with control or Pin1 siRNA. RUNX3 immunoprecipitates were immunoblotted for ubiquitination with anti-ubiquitin antibodies. Levels of RUNX3 and Pin1 are shown in the bottom two panels. (d) MDA-MB-361 cells were transfected with control or Pin1 siRNA. Endogenous RUNX3 immunoprecipitates were immunoblotted for ubiquitination as in (c).

Journal: Oncogene

Article Title: Prolyl isomerase Pin1 down-regulates tumor suppressor RUNX3 in breast cancer

doi: 10.1038/onc.2012.178

Figure Lengend Snippet: (a) Pin1 enhances ubiquitination of RUNX3. HEK293T cells were transfected with indicated combinations of plasmids expressing Flag-RUNX3, HA-ubiquitin, Myc-Pin1 or Myc-Pin1-C115A. RUNX3 immunoprecipitates were immunoblotted for ubiquitination with anti-HA antibodies. Levels of RUNX3 and Pin1 are shown in the lower two panels. (b) Interaction between Pin1 and RUNX3 is important for the Pin1-induced ubiquitination of RUNX3. HEK293T cells were transfected with a combination of Flag-RUNX3 or Flag-RUNX3-4A, HA-ubiquitin and Myc-Pin1 as indicated. RUNX3 ubiquitination was detected as in (a). (c) Down-regulation of Pin1 decreases the ubiquitination of RUNX3. RUNX3-MCF-7 cells were transfected with control or Pin1 siRNA. RUNX3 immunoprecipitates were immunoblotted for ubiquitination with anti-ubiquitin antibodies. Levels of RUNX3 and Pin1 are shown in the bottom two panels. (d) MDA-MB-361 cells were transfected with control or Pin1 siRNA. Endogenous RUNX3 immunoprecipitates were immunoblotted for ubiquitination as in (c).

Article Snippet: Antibody against Pin1 was from R&D systems (Minneapolis, MN, USA), and antibody against RUNX3 was from Abcam (Cambridge, MA, USA).

Techniques: Ubiquitin Proteomics, Transfection, Expressing, Control

Journal: Brain and behavior

Article Title: Upregulation of Pin1 contributes to alleviation of cognitive dysfunction in diabetic mice.

doi: 10.1002/brb3.3336

Figure Lengend Snippet: FIGURE 1 The body weight (a) and blood glucose levels (b) of C57BL/6 mice were measured once a week. Blood samples were collected from the tails of mice under fasting conditions. Body weight was measured in grams, and blood glucose levels were measured using a blood glucose meter. The data are expressed as the mean ± SEM (n = 10). CON, normal control group; DB, diabetic group; DB + 0, sham treatment group; DB + Pin1, experimental group.

Article Snippet: The specific primary antibodies included a rabbit polyclonal antibody against Pin1 (1:1000, Proteintech, 10495-1-AP), a rabbit polyclonal antibody against phospho-tau (Thr231 1:800, AF3147), a rabbit polyclonal antibody against phospho-GSK-3β (Ser9 1:800, AF2016), a rabbit polyclonal antibody against phospho-AKT (Ser473 1:800 ImmunoWay Biotechnology, Inc, YT0176), a rabbit polyclonal antibody against phospho-IRS1 (Ser307 1:800 ImmunoWay Biotechnology, Inc), and a rabbit polyclonal antibody against GAPDH (1:2000, AF7021).

Techniques: Control

FIGURE 2 The Morris water maze (MWM) test was performed before intrahippocampal injection surgery and 1 month after surgery in adult male mice. Learning acquisition curve of adult male mice from 1 month after surgery showing the effects of streptozotocin (STZ) and the protective effects of Pin1 on the mean escape time to reach the hidden platform (escape latency) over consecutive trials in the MWM test. In the probe trials, the number of platform crossings (c) was recorded. The data are expressed as the mean ± SEM (n = 7–10 per group). Statistical analysis was performed using one-way analysis of variance followed by Tukey’s HSD test: (b, c) (*p < .05; **p < .01; ***p < .001). CON, normal control group; DB, diabetic group; DB + 0, sham treatment group; DB + Pin1, experimental group.

Journal: Brain and behavior

Article Title: Upregulation of Pin1 contributes to alleviation of cognitive dysfunction in diabetic mice.

doi: 10.1002/brb3.3336

Figure Lengend Snippet: FIGURE 2 The Morris water maze (MWM) test was performed before intrahippocampal injection surgery and 1 month after surgery in adult male mice. Learning acquisition curve of adult male mice from 1 month after surgery showing the effects of streptozotocin (STZ) and the protective effects of Pin1 on the mean escape time to reach the hidden platform (escape latency) over consecutive trials in the MWM test. In the probe trials, the number of platform crossings (c) was recorded. The data are expressed as the mean ± SEM (n = 7–10 per group). Statistical analysis was performed using one-way analysis of variance followed by Tukey’s HSD test: (b, c) (*p < .05; **p < .01; ***p < .001). CON, normal control group; DB, diabetic group; DB + 0, sham treatment group; DB + Pin1, experimental group.

Techniques: Injection, Control

FIGURE 3 Pin1 mRNA (a) was measured by real-time qRT-PCR, and the protein levels of Pin1 (b), p-AKT (C), p-GSK-3β (d), p-IRS1 (e), and p-Tau (f) were measured by western blotting. The results of three independent experiments were quantified, and the histogram shows the average mRNA or protein expression in each group. The data are expressed as the mean ± SEM (n = 3). Statistical analysis was performed using one-way analysis of variance followed by Tukey’s HSD test (c–f) or Dunnett’s T3 test (A and B) (*p < .05; **p < .01; ***p < .001). CON, normal control group; DB, diabetic group; DB + 0, sham treatment group; DB + Pin1, experimental group.

Journal: Brain and behavior

Article Title: Upregulation of Pin1 contributes to alleviation of cognitive dysfunction in diabetic mice.

doi: 10.1002/brb3.3336

Figure Lengend Snippet: FIGURE 3 Pin1 mRNA (a) was measured by real-time qRT-PCR, and the protein levels of Pin1 (b), p-AKT (C), p-GSK-3β (d), p-IRS1 (e), and p-Tau (f) were measured by western blotting. The results of three independent experiments were quantified, and the histogram shows the average mRNA or protein expression in each group. The data are expressed as the mean ± SEM (n = 3). Statistical analysis was performed using one-way analysis of variance followed by Tukey’s HSD test (c–f) or Dunnett’s T3 test (A and B) (*p < .05; **p < .01; ***p < .001). CON, normal control group; DB, diabetic group; DB + 0, sham treatment group; DB + Pin1, experimental group.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Control

FIGURE 5 Specimens of the CA1 region of the mouse hippocampus were prepared for electron microscopy specimens. The arrow points to a morphologically intact synapse. In the diabetic group, hyperglycemia resulted in significant decreases in synaptic density and axonal swelling after the injection of streptozotocin. The spine density was determined by magnifying the image 8000 times, and the data are expressed as the mean ± SEM (n = 3). Statistical analysis was performed using one-way analysis of variance followed by Tukey’s HSD test (4) (*p < .05; **p < .01; ***p < .001). CON, normal control group; DB, diabetic group; DB + 0, sham treatment group; DB + Pin1, experimental group.

Journal: Brain and behavior

Article Title: Upregulation of Pin1 contributes to alleviation of cognitive dysfunction in diabetic mice.

doi: 10.1002/brb3.3336

Figure Lengend Snippet: FIGURE 5 Specimens of the CA1 region of the mouse hippocampus were prepared for electron microscopy specimens. The arrow points to a morphologically intact synapse. In the diabetic group, hyperglycemia resulted in significant decreases in synaptic density and axonal swelling after the injection of streptozotocin. The spine density was determined by magnifying the image 8000 times, and the data are expressed as the mean ± SEM (n = 3). Statistical analysis was performed using one-way analysis of variance followed by Tukey’s HSD test (4) (*p < .05; **p < .01; ***p < .001). CON, normal control group; DB, diabetic group; DB + 0, sham treatment group; DB + Pin1, experimental group.

Techniques: Electron Microscopy, Injection, Control

FIGURE 4 Compared with those in the DB group, DB + Pin1 group and CON group, hippocampal neurons in the DB group showed increased senescence, a decrease in number, and a disordered arrangement. The neurons in the DB + Pin1 group were more neatly arranged than those in the DB and DB + 0 groups .The mean Pin1 OD was determined by ImageJ (V1.8.0.112), and the data are expressed as the mean ± SEM (n = 3). Statistical analysis was performed using one-way analysis of variance followed by Tukey’s HSD test (5) (*p < .05; **p < .01; ***p < .001). CON, normal control group; DB, diabetic group; DB + 0, sham treatment group; DB + Pin1, experimental group.

Journal: Brain and behavior

Article Title: Upregulation of Pin1 contributes to alleviation of cognitive dysfunction in diabetic mice.

doi: 10.1002/brb3.3336

Figure Lengend Snippet: FIGURE 4 Compared with those in the DB group, DB + Pin1 group and CON group, hippocampal neurons in the DB group showed increased senescence, a decrease in number, and a disordered arrangement. The neurons in the DB + Pin1 group were more neatly arranged than those in the DB and DB + 0 groups .The mean Pin1 OD was determined by ImageJ (V1.8.0.112), and the data are expressed as the mean ± SEM (n = 3). Statistical analysis was performed using one-way analysis of variance followed by Tukey’s HSD test (5) (*p < .05; **p < .01; ***p < .001). CON, normal control group; DB, diabetic group; DB + 0, sham treatment group; DB + Pin1, experimental group.

Techniques: Control

FIGURE 6 In our study, targeted enhancement of Pin1 expression in the hippocampus of diabetic mice led to upregulation of p-IRS-1, p-AKT, and p-GSK-3β expression, downregulation of p-Tau expression, inactivation of p-GSK-3β, and subsequently reduced hyperphosphorylation of Tau, resulting in decreased neurotoxicity.

Journal: Brain and behavior

Article Title: Upregulation of Pin1 contributes to alleviation of cognitive dysfunction in diabetic mice.

doi: 10.1002/brb3.3336

Figure Lengend Snippet: FIGURE 6 In our study, targeted enhancement of Pin1 expression in the hippocampus of diabetic mice led to upregulation of p-IRS-1, p-AKT, and p-GSK-3β expression, downregulation of p-Tau expression, inactivation of p-GSK-3β, and subsequently reduced hyperphosphorylation of Tau, resulting in decreased neurotoxicity.

Techniques: Expressing

$A) In vitro GST pull down assay. The cell lysates were incubated with GST or GST-PIN1 fusion protein, followed by addition of the GST beads. The precipitates were immunoblotted with anti-HIF-1α to show the bound HIF-1α and reprobed with anti-GST to show the precipitated GST and GST-PIN1. B) Interaction of HIF-1α and PIN1 in HCT116 cells. Cells were transfected with Flag-tagged HIF-1α and HA-PIN1, and stimulated with hypoxia for 4 h. The cell lysates were immunoprecipitated with anti-Flag antibody, and the precipitates were fractionated by SDS-polyacrylamide gel electrophoresis and blotted with anti-HA antibody. C) Association of endogenous PIN1 and HIF-1α. HCT116 cells were subjected to hypoxia for 4 h. Cell lysates were incubated with either normal IgG or anti-PIN1 as labeled and blotted with anti HIF-1α. D) Binding of HIF-1α and PIN1 in situ . The HCT116 cells were incubated under normoxia or hypoxia. Interaction of HIF-1α and PIN1 was visualized by Duolink analysis. HIF-1α and PIN1 were co-labeled with antibodies. Nuclei were counter stained with DAPI (blue). Scale bar, 20 μm. E) ITC indicates that PiB binds PIN1. Heat evolution as a function of adding increasing amounts of PiB to GST-PIN1. The heats of dilution were measured separately, and found to be <1% of the signal at the start of titration. Fitting the ITC data with Microcal analysis launcher software indicates that PiB binds to GST-PIN1. Values shown on the figure are from the fit of the displayed dataset. K = 2.11E4 ± 4.46E3M -1 , N = 1.56 ± 0.0451 sites, ΔH = 3.977 ± 0.2676 Kcal/mol, ΔS = 33.1 cal/mol/deg. F) In vitro GST pull down assay. The cell lysates were incubated with GST or GST-PIN1 fusion protein, followed by treating with PiB and addition of the GST beads. The precipitates were immunoblotted with anti-HIF-1α to show the bound HIF-1α and reprobed with anti-GST to show the precipitated GST and GST-PIN1. G) Binding of HIF-1α and inactivated PIN1 in situ . HCT116 cells were incubated with or without PiB (20 μM). Interaction of HIF-1α and inactivated PIN1 was visualized by Duolink analysis. HIF-1α and PIN1 were co-labeled with antibodies. Nuclei were counter stained with DAPI (blue). H) CIP (50 U) was added to the supernatants at 30°C for the indicated time periods. Following incubation, GST or GST-PIN1 proteins were incubated with the supernatants for 4 h and then were pulled down with GST beads. Following incubation, reactions were stopped by the addition of SDS sample buffer, followed by SDS-PAGE. I) HA-HIF-1α proteins were purified using a commercially available kit. The proteins were treated with or without lamda phosphatase for 1 h at 30°C. Then GST-PIN1 proteins were incubated with purified HA-HIF-1α proteins and were pulled down with GST beads. The proteins were resolved in SDS-polyacrylamide gels and detected by immunoblotting. J) Cells were transfected with scrambled siRNA as a negative control or PIN1-siRNA for 72 h and treated with hypoxia for 4 h. Cell lysates were incubated with either normal IgG or anti-MPM2 as labeled and blotted with anti-PIN1. K) HA-HIF-1α, HA-HIF-1α S402/564A , and HA- HIF-1α S641/643A were transfected in HCT116 cells. Whole cell extracts of HCT116 cells were prepared for pull down assays with GST-PIN1 proteins. The pull downed fractions were s with anti-HA antibody.$

Journal: PLoS ONE

Article Title: Peptidyl Prolyl Isomerase PIN1 Directly Binds to and Stabilizes Hypoxia-Inducible Factor-1α

doi: 10.1371/journal.pone.0147038

Figure Lengend Snippet: A) In vitro GST pull down assay. The cell lysates were incubated with GST or GST-PIN1 fusion protein, followed by addition of the GST beads. The precipitates were immunoblotted with anti-HIF-1α to show the bound HIF-1α and reprobed with anti-GST to show the precipitated GST and GST-PIN1. B) Interaction of HIF-1α and PIN1 in HCT116 cells. Cells were transfected with Flag-tagged HIF-1α and HA-PIN1, and stimulated with hypoxia for 4 h. The cell lysates were immunoprecipitated with anti-Flag antibody, and the precipitates were fractionated by SDS-polyacrylamide gel electrophoresis and blotted with anti-HA antibody. C) Association of endogenous PIN1 and HIF-1α. HCT116 cells were subjected to hypoxia for 4 h. Cell lysates were incubated with either normal IgG or anti-PIN1 as labeled and blotted with anti HIF-1α. D) Binding of HIF-1α and PIN1 in situ . The HCT116 cells were incubated under normoxia or hypoxia. Interaction of HIF-1α and PIN1 was visualized by Duolink analysis. HIF-1α and PIN1 were co-labeled with antibodies. Nuclei were counter stained with DAPI (blue). Scale bar, 20 μm. E) ITC indicates that PiB binds PIN1. Heat evolution as a function of adding increasing amounts of PiB to GST-PIN1. The heats of dilution were measured separately, and found to be <1% of the signal at the start of titration. Fitting the ITC data with Microcal analysis launcher software indicates that PiB binds to GST-PIN1. Values shown on the figure are from the fit of the displayed dataset. K = 2.11E4 ± 4.46E3M -1 , N = 1.56 ± 0.0451 sites, ΔH = 3.977 ± 0.2676 Kcal/mol, ΔS = 33.1 cal/mol/deg. F) In vitro GST pull down assay. The cell lysates were incubated with GST or GST-PIN1 fusion protein, followed by treating with PiB and addition of the GST beads. The precipitates were immunoblotted with anti-HIF-1α to show the bound HIF-1α and reprobed with anti-GST to show the precipitated GST and GST-PIN1. G) Binding of HIF-1α and inactivated PIN1 in situ . HCT116 cells were incubated with or without PiB (20 μM). Interaction of HIF-1α and inactivated PIN1 was visualized by Duolink analysis. HIF-1α and PIN1 were co-labeled with antibodies. Nuclei were counter stained with DAPI (blue). H) CIP (50 U) was added to the supernatants at 30°C for the indicated time periods. Following incubation, GST or GST-PIN1 proteins were incubated with the supernatants for 4 h and then were pulled down with GST beads. Following incubation, reactions were stopped by the addition of SDS sample buffer, followed by SDS-PAGE. I) HA-HIF-1α proteins were purified using a commercially available kit. The proteins were treated with or without lamda phosphatase for 1 h at 30°C. Then GST-PIN1 proteins were incubated with purified HA-HIF-1α proteins and were pulled down with GST beads. The proteins were resolved in SDS-polyacrylamide gels and detected by immunoblotting. J) Cells were transfected with scrambled siRNA as a negative control or PIN1-siRNA for 72 h and treated with hypoxia for 4 h. Cell lysates were incubated with either normal IgG or anti-MPM2 as labeled and blotted with anti-PIN1. K) HA-HIF-1α, HA-HIF-1α S402/564A , and HA- HIF-1α S641/643A were transfected in HCT116 cells. Whole cell extracts of HCT116 cells were prepared for pull down assays with GST-PIN1 proteins. The pull downed fractions were s with anti-HA antibody.

Article Snippet: Antibodies against phospho-PIN1 and HA-tag were products of Cell Signaling Technology (Beverly, MA, USA).

Techniques: In Vitro, Pull Down Assay, Incubation, Transfection, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Labeling, Binding Assay, In Situ, Staining, Titration, Software, SDS Page, Purification, Western Blot, Negative Control

A) Silencing of PIN1 expression via RNAi reduces the protein levels of HIF-1α in HCT116 cells. Western blot analysis was performed with 50 μg total cell lysate from HCT116 cells transfected with control or PIN1 si-RNA. B) mRNA levels of HIF-1α and PIN1 were determined by RT-PCR analysis at the indicated time points under hypoxic conditions. GAPDH was used as internal controls. C) The PIN1 inhibitor, PiB, reduces the protein levels of HIF-1α in HCT116 cells. HCT116 cells were mock-treated or treated with PiB and harvested 8 h after treatment for the analysis of the HIF-1α and PIN1 levels. D) HCT116 cells were mock-treated or treated with PiB and harvested 8 h later. mRNA levels of HIF-1α and PIN1 were determined by RT-PCR analysis at the indicated time points under hypoxic conditions. GAPDH was used as internal controls. E) ATRA decreases the expression of HIF-1α and PIN1 in HCT116/5xHRE-ODD-luc cells in dose-dependent manner. F) The luciferase activity of HRE in HCT116/5xHRE-ODD-luc cells treated ATRA was reduced in dose-dependent manner. G) Nuclear co-localization of HIF-1α and PIN1 was determined by immunocytochemistry. HCT116 cells plated on the chamber slide were transfected PIN1 si-RNA or treated PiB and grown at 1% O 2 in a hypoxia chamber for additional 4 h. The samples were co-incubated with anti HIF-1α and PIN1 antibodies and then co-incubated with FITC-conjugated secondary antibodies. Lastly, samples were stained with DAPI. The signals were detected using a confocal microscope or a fluorescent microscope. Scale bar, 10 μm.

Journal: PLoS ONE

Article Title: Peptidyl Prolyl Isomerase PIN1 Directly Binds to and Stabilizes Hypoxia-Inducible Factor-1α

doi: 10.1371/journal.pone.0147038

Figure Lengend Snippet: A) Silencing of PIN1 expression via RNAi reduces the protein levels of HIF-1α in HCT116 cells. Western blot analysis was performed with 50 μg total cell lysate from HCT116 cells transfected with control or PIN1 si-RNA. B) mRNA levels of HIF-1α and PIN1 were determined by RT-PCR analysis at the indicated time points under hypoxic conditions. GAPDH was used as internal controls. C) The PIN1 inhibitor, PiB, reduces the protein levels of HIF-1α in HCT116 cells. HCT116 cells were mock-treated or treated with PiB and harvested 8 h after treatment for the analysis of the HIF-1α and PIN1 levels. D) HCT116 cells were mock-treated or treated with PiB and harvested 8 h later. mRNA levels of HIF-1α and PIN1 were determined by RT-PCR analysis at the indicated time points under hypoxic conditions. GAPDH was used as internal controls. E) ATRA decreases the expression of HIF-1α and PIN1 in HCT116/5xHRE-ODD-luc cells in dose-dependent manner. F) The luciferase activity of HRE in HCT116/5xHRE-ODD-luc cells treated ATRA was reduced in dose-dependent manner. G) Nuclear co-localization of HIF-1α and PIN1 was determined by immunocytochemistry. HCT116 cells plated on the chamber slide were transfected PIN1 si-RNA or treated PiB and grown at 1% O 2 in a hypoxia chamber for additional 4 h. The samples were co-incubated with anti HIF-1α and PIN1 antibodies and then co-incubated with FITC-conjugated secondary antibodies. Lastly, samples were stained with DAPI. The signals were detected using a confocal microscope or a fluorescent microscope. Scale bar, 10 μm.

Article Snippet: Antibodies against phospho-PIN1 and HA-tag were products of Cell Signaling Technology (Beverly, MA, USA).

Techniques: Expressing, Western Blot, Transfection, Control, Reverse Transcription Polymerase Chain Reaction, Luciferase, Activity Assay, Immunocytochemistry, Incubation, Staining, Microscopy

A) Lysates of cells were transfected with DNA-based siRNA expression vectors for 72 h, then exposed to hypoxia for 4 h, and then treated with CHX (10 μM) for the indicated time periods under hypoxic condition were used for Western blotting analysis with anti-HIF-1α and anti-PIN1. Relative amounts of the HIF-1α signals, normalized to the values obtained in the absence of CHX, were plotted and used to calculate half-lives. Relative levels of the HIF-1α signals were plotted and used to calculate half-lives. PIN1 depletion significantly reduced the stability of HIF-1α half-life. B) PIN1 induces the conformational change of HIF-1α. Limited proteolysis of purified HA-HIF-1α derived from HCT116 cells transfected GFP-PIN1 or GFP. The purified HA-HIF-1α was subjected to partial chymotrypsin digestion and analyzed by Western blotting.

Journal: PLoS ONE

Article Title: Peptidyl Prolyl Isomerase PIN1 Directly Binds to and Stabilizes Hypoxia-Inducible Factor-1α

doi: 10.1371/journal.pone.0147038

Figure Lengend Snippet: A) Lysates of cells were transfected with DNA-based siRNA expression vectors for 72 h, then exposed to hypoxia for 4 h, and then treated with CHX (10 μM) for the indicated time periods under hypoxic condition were used for Western blotting analysis with anti-HIF-1α and anti-PIN1. Relative amounts of the HIF-1α signals, normalized to the values obtained in the absence of CHX, were plotted and used to calculate half-lives. Relative levels of the HIF-1α signals were plotted and used to calculate half-lives. PIN1 depletion significantly reduced the stability of HIF-1α half-life. B) PIN1 induces the conformational change of HIF-1α. Limited proteolysis of purified HA-HIF-1α derived from HCT116 cells transfected GFP-PIN1 or GFP. The purified HA-HIF-1α was subjected to partial chymotrypsin digestion and analyzed by Western blotting.

Article Snippet: Antibodies against phospho-PIN1 and HA-tag were products of Cell Signaling Technology (Beverly, MA, USA).

Techniques: Transfection, Expressing, Western Blot, Purification, Derivative Assay

A) Effect of PIN1 knockdown on EPO-HRE-luciferase reporter activity under normoxia or hypoxia for 24 h. HCT116 cells were transfected with control or PIN1 si-RNA for 72 h and then were transfected with pGL2-luc and EPO-HRE-luc for another 24 h. Cells were lysed to analyze luciferase activities, which were normalized against β-galactosidase activities. B, C) In vitro HIF-1α bioluminescence assay. 3 x 10 5 HCT116/5xHRE-ODD-luc cells were transfected with control or PIN1 si-RNA for 24 h (B) or treated with PiB (C) and harvested 8 h after treatment. The cells were incubated in each well of a 96-well-dish in a hypoxia chamber (1% O 2 ) for 4 h before the medium was removed, washed and replaced with 1 ml PBS. Immediately after 100 μl luciferin was added into each well, ROIs were acquired with an array of exposure times (1, 30, 60, and 180 s). D) PIN1 regulates transcriptional activity of HIF-1 during hypoxia-mimic conditions. Hypoxia was induced by DFO (400 μM). HCT116/5xHRE-luc cells were treated with DFO only, DFO plus PiB (20 μM), or PiB (20 μM) alone for 8 h. ROIs were analysed by bioluminescence imaging from HCT116/5xHRE-luc cells after various treatments.

Journal: PLoS ONE

Article Title: Peptidyl Prolyl Isomerase PIN1 Directly Binds to and Stabilizes Hypoxia-Inducible Factor-1α

doi: 10.1371/journal.pone.0147038

Figure Lengend Snippet: A) Effect of PIN1 knockdown on EPO-HRE-luciferase reporter activity under normoxia or hypoxia for 24 h. HCT116 cells were transfected with control or PIN1 si-RNA for 72 h and then were transfected with pGL2-luc and EPO-HRE-luc for another 24 h. Cells were lysed to analyze luciferase activities, which were normalized against β-galactosidase activities. B, C) In vitro HIF-1α bioluminescence assay. 3 x 10 5 HCT116/5xHRE-ODD-luc cells were transfected with control or PIN1 si-RNA for 24 h (B) or treated with PiB (C) and harvested 8 h after treatment. The cells were incubated in each well of a 96-well-dish in a hypoxia chamber (1% O 2 ) for 4 h before the medium was removed, washed and replaced with 1 ml PBS. Immediately after 100 μl luciferin was added into each well, ROIs were acquired with an array of exposure times (1, 30, 60, and 180 s). D) PIN1 regulates transcriptional activity of HIF-1 during hypoxia-mimic conditions. Hypoxia was induced by DFO (400 μM). HCT116/5xHRE-luc cells were treated with DFO only, DFO plus PiB (20 μM), or PiB (20 μM) alone for 8 h. ROIs were analysed by bioluminescence imaging from HCT116/5xHRE-luc cells after various treatments.

Article Snippet: Antibodies against phospho-PIN1 and HA-tag were products of Cell Signaling Technology (Beverly, MA, USA).

Techniques: Knockdown, Luciferase, Activity Assay, Transfection, Control, In Vitro, ATP Bioluminescent Assay, Incubation, Imaging

A) The HCT116 cells were transfected with control or PIN1 si-RNA under hypoxia. Western blot analysis was performed with anti-VEGF-A. B) For analysis of VEGF-A mRNA, RT-PCR was performed with 1 μg total RNA prepared from HCT116 cells transfected with control or PIN1 si-RNA under hypoxia. C) The PIN1 inhibitor, PiB, reduces the protein levels of HIF-1α in HCT116 cells. The cells were mock-treated or treated with PiB and harvested 8 h. Western blot analysis was conducted as described Fig 5A . D) For the analysis of VEGF-A mRNA, HCT116 cells were mock-treated or treated with PiB and harvested 8 h. RT-PCR analysis was performed as described Fig 5B .

Journal: PLoS ONE

Article Title: Peptidyl Prolyl Isomerase PIN1 Directly Binds to and Stabilizes Hypoxia-Inducible Factor-1α

doi: 10.1371/journal.pone.0147038

Figure Lengend Snippet: A) The HCT116 cells were transfected with control or PIN1 si-RNA under hypoxia. Western blot analysis was performed with anti-VEGF-A. B) For analysis of VEGF-A mRNA, RT-PCR was performed with 1 μg total RNA prepared from HCT116 cells transfected with control or PIN1 si-RNA under hypoxia. C) The PIN1 inhibitor, PiB, reduces the protein levels of HIF-1α in HCT116 cells. The cells were mock-treated or treated with PiB and harvested 8 h. Western blot analysis was conducted as described Fig 5A . D) For the analysis of VEGF-A mRNA, HCT116 cells were mock-treated or treated with PiB and harvested 8 h. RT-PCR analysis was performed as described Fig 5B .

Article Snippet: Antibodies against phospho-PIN1 and HA-tag were products of Cell Signaling Technology (Beverly, MA, USA).

Techniques: Transfection, Control, Western Blot, Reverse Transcription Polymerase Chain Reaction

A) HUVEC were transfected with control or PIN1 si-RNA for 24 h and then were cultured in Matrigel in supplemented media. B) HUVEC were suspended in Matrigel with VEGF in the presence or absence of different concentrations of PiB and maintained for 8 h. Quantification of area of cell alignment was determined by image J software (**P < 0.001). C) Effect of PiB on aortic ring sprouting. Aortic rings were treated with VEGF (100 ng) and bFGF (100 ng) in the presence or absence of different concentrations of PiB for 9 days. Quantification of area of cell alignment was determined by image J software (*P < 0.05, **P < 0.001).

Journal: PLoS ONE

Article Title: Peptidyl Prolyl Isomerase PIN1 Directly Binds to and Stabilizes Hypoxia-Inducible Factor-1α

doi: 10.1371/journal.pone.0147038

Figure Lengend Snippet: A) HUVEC were transfected with control or PIN1 si-RNA for 24 h and then were cultured in Matrigel in supplemented media. B) HUVEC were suspended in Matrigel with VEGF in the presence or absence of different concentrations of PiB and maintained for 8 h. Quantification of area of cell alignment was determined by image J software (**P < 0.001). C) Effect of PiB on aortic ring sprouting. Aortic rings were treated with VEGF (100 ng) and bFGF (100 ng) in the presence or absence of different concentrations of PiB for 9 days. Quantification of area of cell alignment was determined by image J software (*P < 0.05, **P < 0.001).

Article Snippet: Antibodies against phospho-PIN1 and HA-tag were products of Cell Signaling Technology (Beverly, MA, USA).

Techniques: Transfection, Control, Cell Culture, Software

A) Silencing of PIN1 via sh-RNA lentiviral vector reduces the protein levels of HIF-1α in HCT116/5xHRE-ODD-luc cells. Western blot analysis was performed with 50 μg total cell lysate form HCT116/5xHRE-ODD-luc cells infected with control or sh-PIN1 virus. B) The luciferase activity of HRE in HCT116/5xHRE-ODD-luc cells infected with sh-PIN1 virus was reduced compared to control cells. C) HCT116/5xHRE-ODD-luc cells were infected with sh-PIN1 or control virus. The cells were subcutaneously injected into male BALB/c nu/nu mice (n = 6). Hb levels of Matrigel plugs from excised tumors after autopsy. Hb levels in the supernatant were quantified with the Drabkin’s reagent kit. The data are means ± SD from replicate experiments (n = 6) (**P < 0.001). D) PiB inhibited tumor angiogenesis in nude mice. Growth factor-reduced BD Matrigel containing HCT116 cells and indicated concentrations of PiB were subcutaneously injected into male BALB/c nu/nu mice as described in Materials and Methods. The photos are representative Matrigel plugs. Hb levels of Matrigel plugs. Hb levels in the supernatant were quantified with the Drabkin’s reagent kit. The data are means ± SD from replicate experiments (n = 4) (*P < 0.05, **P < 0.001).

Journal: PLoS ONE

Article Title: Peptidyl Prolyl Isomerase PIN1 Directly Binds to and Stabilizes Hypoxia-Inducible Factor-1α

doi: 10.1371/journal.pone.0147038

Figure Lengend Snippet: A) Silencing of PIN1 via sh-RNA lentiviral vector reduces the protein levels of HIF-1α in HCT116/5xHRE-ODD-luc cells. Western blot analysis was performed with 50 μg total cell lysate form HCT116/5xHRE-ODD-luc cells infected with control or sh-PIN1 virus. B) The luciferase activity of HRE in HCT116/5xHRE-ODD-luc cells infected with sh-PIN1 virus was reduced compared to control cells. C) HCT116/5xHRE-ODD-luc cells were infected with sh-PIN1 or control virus. The cells were subcutaneously injected into male BALB/c nu/nu mice (n = 6). Hb levels of Matrigel plugs from excised tumors after autopsy. Hb levels in the supernatant were quantified with the Drabkin’s reagent kit. The data are means ± SD from replicate experiments (n = 6) (**P < 0.001). D) PiB inhibited tumor angiogenesis in nude mice. Growth factor-reduced BD Matrigel containing HCT116 cells and indicated concentrations of PiB were subcutaneously injected into male BALB/c nu/nu mice as described in Materials and Methods. The photos are representative Matrigel plugs. Hb levels of Matrigel plugs. Hb levels in the supernatant were quantified with the Drabkin’s reagent kit. The data are means ± SD from replicate experiments (n = 4) (*P < 0.05, **P < 0.001).

Article Snippet: Antibodies against phospho-PIN1 and HA-tag were products of Cell Signaling Technology (Beverly, MA, USA).

Techniques: Plasmid Preparation, Western Blot, Infection, Control, Virus, Luciferase, Activity Assay, Injection

A) In vivo bioluminescence imaging. Luciferase signals from PiB-treated tumor were lower than those from non-treated tumor. B) From ROIs analysis of in vivo bioluminescence imaging, signals in tumors from PiB treated mice were significantly reduced compare to non-treated tumor (P = 0.0016). C) The size of PiB-treated tumor was significantly decreased (P = 0.0212) compared to non-treated tumors. D) Bioluminescence images from excised tumors after autopsy showed that luciferase signals in tumor tissues from PiB-treated animals were lower than that of control tumor, indicating reduced activation of HRE. E) Immunohistochemistry data showed that tumors from PiB-treated mice express less luciferase, VEGF, and CD31 compared to non-treated tumors. F) Co-localization of PIN1 and HIF-1α was observed using confocal fluorescence microscopy. Scale bar, 20 μm.

Journal: PLoS ONE

Article Title: Peptidyl Prolyl Isomerase PIN1 Directly Binds to and Stabilizes Hypoxia-Inducible Factor-1α

doi: 10.1371/journal.pone.0147038

Figure Lengend Snippet: A) In vivo bioluminescence imaging. Luciferase signals from PiB-treated tumor were lower than those from non-treated tumor. B) From ROIs analysis of in vivo bioluminescence imaging, signals in tumors from PiB treated mice were significantly reduced compare to non-treated tumor (P = 0.0016). C) The size of PiB-treated tumor was significantly decreased (P = 0.0212) compared to non-treated tumors. D) Bioluminescence images from excised tumors after autopsy showed that luciferase signals in tumor tissues from PiB-treated animals were lower than that of control tumor, indicating reduced activation of HRE. E) Immunohistochemistry data showed that tumors from PiB-treated mice express less luciferase, VEGF, and CD31 compared to non-treated tumors. F) Co-localization of PIN1 and HIF-1α was observed using confocal fluorescence microscopy. Scale bar, 20 μm.

Article Snippet: Antibodies against phospho-PIN1 and HA-tag were products of Cell Signaling Technology (Beverly, MA, USA).

Techniques: In Vivo, Imaging, Luciferase, Control, Activation Assay, Immunohistochemistry, Fluorescence, Microscopy

A) In vivo bioluminescence imaging. HCT116/5xHRE-ODD-luc cells were infected with sh-PIN1 or control virus. The cells were subcutaneously injected into male BALB/c nu/nu mice (n = 6). Luciferase signals from sh-PIN1 infected tumor were lower lower than those from sh-mock infected tumor. B) Signals in tumors infected sh-PIN1 were significantly reduced compare to tumors of sh-mock from ROIs analysis of in vivo bioluminescence imaging. C) The volume of tumors infected sh-PIN1 was significantly decreased compare to control tumors.

Journal: PLoS ONE

Article Title: Peptidyl Prolyl Isomerase PIN1 Directly Binds to and Stabilizes Hypoxia-Inducible Factor-1α

doi: 10.1371/journal.pone.0147038

Figure Lengend Snippet: A) In vivo bioluminescence imaging. HCT116/5xHRE-ODD-luc cells were infected with sh-PIN1 or control virus. The cells were subcutaneously injected into male BALB/c nu/nu mice (n = 6). Luciferase signals from sh-PIN1 infected tumor were lower lower than those from sh-mock infected tumor. B) Signals in tumors infected sh-PIN1 were significantly reduced compare to tumors of sh-mock from ROIs analysis of in vivo bioluminescence imaging. C) The volume of tumors infected sh-PIN1 was significantly decreased compare to control tumors.

Article Snippet: Antibodies against phospho-PIN1 and HA-tag were products of Cell Signaling Technology (Beverly, MA, USA).

Techniques: In Vivo, Imaging, Infection, Control, Virus, Injection, Luciferase

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