Journal: Current Issues in Molecular Biology

Article Title: The Role of WNT3A Protein and Gene Variants in Allergic Rhinitis: A Case-Control Study

doi: 10.3390/cimb46090565

Figure Lengend Snippet: Comparison of serum WNT3A levels between the patient and control groups. (**** p < 0.0001).

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) analysis was performed according to the manufacturer’s instructions using the Human Protein WNT3A ELISA kit (BT Lab, Jiaxing, China).

Techniques: Comparison, Control

Journal: Current Issues in Molecular Biology

Article Title: The Role of WNT3A Protein and Gene Variants in Allergic Rhinitis: A Case-Control Study

doi: 10.3390/cimb46090565

Figure Lengend Snippet: ROC curve of WNT3A protein between control and AR groups. Blue dashed line indicates cut off point. AUC; area under the curve.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) analysis was performed according to the manufacturer’s instructions using the Human Protein WNT3A ELISA kit (BT Lab, Jiaxing, China).

Techniques: Control

Journal: Current Issues in Molecular Biology

Article Title: The Role of WNT3A Protein and Gene Variants in Allergic Rhinitis: A Case-Control Study

doi: 10.3390/cimb46090565

Figure Lengend Snippet: Comparison of serum WNT3A levels according to patients’ disease characteristics.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) analysis was performed according to the manufacturer’s instructions using the Human Protein WNT3A ELISA kit (BT Lab, Jiaxing, China).

Techniques: Comparison

Journal: Current Issues in Molecular Biology

Article Title: The Role of WNT3A Protein and Gene Variants in Allergic Rhinitis: A Case-Control Study

doi: 10.3390/cimb46090565

Figure Lengend Snippet: Comparison of patients’ clinical data based on their genotypes.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) analysis was performed according to the manufacturer’s instructions using the Human Protein WNT3A ELISA kit (BT Lab, Jiaxing, China).

Techniques: Comparison

Journal: Journal of Lipid Research

Article Title: FASN negatively regulates p65 expression by reducing its stability via Thr 254 phosphorylation and isomerization by Pin1

doi: 10.1016/j.jlr.2024.100529

Figure Lengend Snippet: FASN regulation of NF-κB activity. A: Effect of FASN expression on reporter activity driven by NF-κB. Stable M3K/Sh(FASN) and MDA-MB-436/FASN cells with their respective control M3K/Scr and MDA-MB-436/Vec cells were transfected with NF-κB cis-reporter plasmid expressing the firefly luciferase gene along with a pRL-TK renilla luciferase plasmid as a control for transfection efficiency. At 48 h after transfection, dual luciferase activity assay was performed to measure luciferase gene expression as an output of NF-κB activity. B: Immunofluorescence staining of p65 in MDA-MB-436/FASN and its control MDA-MB-436/Vec cells. Scale bar represents 25 μm. C–F: Effect of FASN on TNF-α and PARP1 expression. Stable M3K/Sh(FASN) and MDA-MB-436/FASN cells along with their respective control M3K/Scr and MDA-MB-436/Vec cells were subjected to analyses of TNF-α and PARP1 mRNA using real-time RT-PCR (C, F) or PARP1 protein using Western blot (D, E). E: Quantification of Western blot data shown in D from ≥3 independent experiments (∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001).

Article Snippet: FASN antibody (610,962; 1:1,000 dilution), Dual-Luciferase Assay System, and pCMV3-FLAG-Pin1 plasmid (HG10282-NF) were purchased from BD Biosciences (Franklin Lakes, NJ), Promega Corporation (Madison, WI), and Sino Biological (Wayne, PA), respectively.

Techniques: Activity Assay, Expressing, Transfection, Plasmid Preparation, Luciferase, Immunofluorescence, Staining, Quantitative RT-PCR, Western Blot

Journal: Scientific reports

Article Title: Identification of potential new T cell activation molecules: a Bioinformatic Approach.

doi: 10.1038/s41598-024-73003-9

Figure Lengend Snippet: Fig. 3. Relative expression over time of genes with potential participation in early T-cell activation from 0 to 18 h post activation from data set GSE136625 (A-F). We present the expression of CD69, MDM4, PIN1, SYT10, RND3, IGSF6 at 0, 2, 4, 6 and 18 h showing the induction and eventual decrease near 18 h for most of them, except for Pin1 that seem to continue increasing. And from GSE902 (G-K). We present the expression of CD69, PIN1, PLAGL2, ABCF1 and CD53 at 1, 2, 4, 8 and 18 h in Jurkat cells showing similar patterns.

Article Snippet: Then the cells were washed 3 times with PBS and incubated for 1 h with primary antibody: α-CD69 (1:20, Abcam, Ab181602), α-CD40 (1:20, Abcam, Ab13545), α-SYT10 (1:20, Novus Biologicals, NBP2-85861), α-RND3 (1:50, Abnova, H00000390-MOI), α-DORA (1:20, EuroScientific, DDX0220P-100), α-GAPDH (1:50, Abcam, Ab233396), αCD53 (1:20, Novus biologicals, NB500393), α-PIN1 (1:20, R&D Systems, MAB2294), Scientific Reports | (2024) 14:22219 9| https://doi.org/10.1038/s41598-024-73003-9 α-MDM4 (1:20, Novus Biologicals, NB100556) dissolved in 1X PBS with 2% FBS.

Techniques: Expressing, Activation Assay

Journal: Scientific reports

Article Title: Identification of potential new T cell activation molecules: a Bioinformatic Approach.

doi: 10.1038/s41598-024-73003-9

Figure Lengend Snippet: Fig. 5. Candidate early activation molecules expression. Representative confocal images for the expression of CD69, CD40, IgSF6, Pin1, RND3, Syt10, MDM4 and GAPDH in resting or 4 h stimulated Jurkat cells with anti CD3 plus anti CD28, surface molecules were evaluated under non permeabilized (to detect surface expression) or permeabilized conditions (to detect potential intracellular pools plus surface expression). Bar represents 10 μm. Below quantification of the fluorescence intensity of different number of cells (indicated below each graph) at resting and 4 h after stimulation, unpaired two tailed t test analysis bars correspond to SEM and *p < 0.05, **p < 0.005, **** p < 0.0001.

Article Snippet: Then the cells were washed 3 times with PBS and incubated for 1 h with primary antibody: α-CD69 (1:20, Abcam, Ab181602), α-CD40 (1:20, Abcam, Ab13545), α-SYT10 (1:20, Novus Biologicals, NBP2-85861), α-RND3 (1:50, Abnova, H00000390-MOI), α-DORA (1:20, EuroScientific, DDX0220P-100), α-GAPDH (1:50, Abcam, Ab233396), αCD53 (1:20, Novus biologicals, NB500393), α-PIN1 (1:20, R&D Systems, MAB2294), Scientific Reports | (2024) 14:22219 9| https://doi.org/10.1038/s41598-024-73003-9 α-MDM4 (1:20, Novus Biologicals, NB100556) dissolved in 1X PBS with 2% FBS.

Techniques: Activation Assay, Expressing, Fluorescence, Two Tailed Test

Journal: Scientific reports

Article Title: Identification of potential new T cell activation molecules: a Bioinformatic Approach.

doi: 10.1038/s41598-024-73003-9

Figure Lengend Snippet: Fig. 6. Identification of novel potential activation molecules in primary human T cells. Freshly isolated naïve CD4 T cells were stimulated for 0, 4, 6 and 18 h with anti CD3 plus anti CD28 and processed for immunofluorescence for the expression of CD69, CD40, CD53, IgSF6 (DORA), Syt10, RND3, Pin1 and MDM4, bar correspond to 10 μm. Right panels represent the quantification of 3 independent experiments (1 donor each experiment) with the total number of cells below each graph and time, bars correspond to SEM and **p < 0.01, ***p < 0.001, **** p < 0.0001.

Article Snippet: Then the cells were washed 3 times with PBS and incubated for 1 h with primary antibody: α-CD69 (1:20, Abcam, Ab181602), α-CD40 (1:20, Abcam, Ab13545), α-SYT10 (1:20, Novus Biologicals, NBP2-85861), α-RND3 (1:50, Abnova, H00000390-MOI), α-DORA (1:20, EuroScientific, DDX0220P-100), α-GAPDH (1:50, Abcam, Ab233396), αCD53 (1:20, Novus biologicals, NB500393), α-PIN1 (1:20, R&D Systems, MAB2294), Scientific Reports | (2024) 14:22219 9| https://doi.org/10.1038/s41598-024-73003-9 α-MDM4 (1:20, Novus Biologicals, NB100556) dissolved in 1X PBS with 2% FBS.

Techniques: Activation Assay, Isolation, Immunofluorescence, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Analysis of binding interfaces of the human scaffold protein AXIN1 by peptide microarrays

doi: 10.1074/jbc.RA118.005127

Figure Lengend Snippet: Domain structure of AXIN1 and its interaction partners. A, top, secondary structure prediction of human AXIN1 (hAXIN1) protein performed by PONDR-FIT software (33). Values <0.5 indicate structured protein regions (ordered; in gray color); values >0.5 indicate intrinsic parts lacking secondary structure (disordered; in purple color). RGS and DIX domains are indicated, and the central IDR is depicted in purple. Bottom, overview of binding regions of known selected AXIN1-interacting proteins from various signaling pathways reported in the literature: CK1ϵ, c-Myc, Pin1, and p53, and DVL, respectively. B, the general workflow of a peptide microarray approach used to study protein–protein interactions mediated via IDRs: a glass slide containing immobilized peptides is incubated with the protein of interest followed by incubation with primary antibody against the recombinant protein. The positive spots are then visualized with a fluorescently labeled secondary antibody. See “Experimental procedures” for details.

Article Snippet: Then the experimental slide was incubated overnight with 10 μg/ml recombinant protein, CK1ε (OriGene Technologies, TP302436), p53 (OriGene Technologies, TP300003) Pin1 (OriGene Technologies, TP302543), or c-Myc (Abnova, H00004609-P01), in SmartBlock solution (final volume, 300 μl) at 4 °C.

Techniques: Software, Binding Assay, Protein-Protein interactions, Peptide Microarray, Incubation, Recombinant, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Analysis of binding interfaces of the human scaffold protein AXIN1 by peptide microarrays

doi: 10.1074/jbc.RA118.005127

Figure Lengend Snippet: AXIN1-binding epitopes for CK1ϵ, c-Myc, Pin1, and p53 identified by peptide array approach. The microarray slides with AXIN1 peptide library were incubated with the following purified recombinant proteins: CK1ϵ (A), c-Myc (B), p53 (C), and Pin1 (D). The charts show normalized signal values from single peptide spots, and the dashed line represents the threshold signal intensity. AXIN1 is schematically depicted above the charts with the numbers and sequences of amino acids defining the particular binding sites. The signals from control glass (antibodies only) were subtracted from the experimental values, and the four strongest signals of at least four consecutive (i.e. neighboring and overlapping) peptides higher than 10,000 LUs were considered as candidate binding sites; see “Experimental procedures” and main text for more details. For each protein of interest, the average from two to three replicates is shown. hAXIN1, human AXIN1.

Article Snippet: Then the experimental slide was incubated overnight with 10 μg/ml recombinant protein, CK1ε (OriGene Technologies, TP302436), p53 (OriGene Technologies, TP300003) Pin1 (OriGene Technologies, TP302543), or c-Myc (Abnova, H00004609-P01), in SmartBlock solution (final volume, 300 μl) at 4 °C.

Techniques: Binding Assay, Peptide Microarray, Microarray, Incubation, Purification, Recombinant, Control

Journal: Nature communications

Article Title: Depletion of mitochondrial methionine adenosyltransferase α1 triggers mitochondrial dysfunction in alcohol-associated liver disease.

doi: 10.1038/s41467-022-28201-2

Figure Lengend Snippet: Fig. 2 Alcohol enhances PIN1 and MATα1 interaction in the liver. a Human and mouse MATα1 protein sequences showing that PIN1 binding motif is conserved between species. b IP analysis of human hepatocytes lysate using anti-PIN1 antibody followed by western blot analysis against MATα1. c IP analysis of WT mouse liver hepatocytes lysate with anti-PIN1 (left panel) or anti-MATα1 (right panel) antibody followed by western blot analysis against MATα1 (left panel) or PIN1 (right panel). d IP analysis using PIN1 and MATα1 recombinant proteins and beads conjugated to anti-PIN1 and anti-MATα1 antibodies. IgG was used as a negative control. IP analysis of human normal and end-stage cirrhotic ALD liver lysates (e) (n = 3 normal and n = 5 ALD independent patients. p = 0.044), pair-fed and ethanol-fed mouse livers whole lysates (f, n = 4 pair-fed and n = 4 ethanol-fed independent animals. p = 0.002) and (g, n = 3 pair-fed and n = 3 ethanol-fed independent animals p = 0.027) and cytosolic fractions (h, n = 3 pair-fed and n = 3 ethanol-fed independent animals. p = 0.038), and AML-12 cells treated with ethanol (i and j, n = 3 independent experiments. p = 0.005) using anti-PIN1 or anti- MATα1 antibodies followed by western blot analysis against MATα1 or PIN1. Densitometry analysis are shown; ethanol-fed vs pair-fed; and ethanol vs control. Results are shown as mean ± SEM. *p < 0.05. Statistical significance was determined by using two-tailed, one-sample t-tests. Source data are provided as a Source data file. ALD alcohol-associated liver disease, IP immunoprecipitation, r recombinant.

Article Snippet: For PIN1 knockdown 50 nM siRNA against human PIN1 (SR303529, Origene) or mouse Pin1 (SR403748, Origene) or equivalent scramble control (SC) were delivered into HepG2 or AML-12 cells by Lipofectamine RNAiMAX (Life Technologies) for 48 h following the manufacturer’s protocol.

Techniques: Binding Assay, Western Blot, Recombinant, Negative Control, Control, Two Tailed Test, Immunoprecipitation

Journal: Nature communications

Article Title: Depletion of mitochondrial methionine adenosyltransferase α1 triggers mitochondrial dysfunction in alcohol-associated liver disease.

doi: 10.1038/s41467-022-28201-2

Figure Lengend Snippet: Fig. 4 Alcohol-induced MATα1 phosphorylation at Ser114 is required for PIN1 binding. a IP analysis of human normal and AH liver lysates, pair-fed and ethanol-fed mouse livers, and AML-12 cells treated with ethanol using anti-phosphoserine antibody followed by western blot analysis against MATα1 (n = 3 independent samples/experiments, p = 0.001 for AH vs normal; p = 0.021 ethanol-fed vs pair-fed; and p = 0.008 ethanol vs control AML-12). b Phos-Tag gels of phosphorylated MATα1 in cytosolic and mitochondrial fractions of AML-12 cells treated with ethanol. Unphosphorylated and phosphorylated recombinant human MATα1 protein is shown as a control (left). c Area under the curve of the phosphorylated peptide which corresponds to MATα1 Ser114 and its unmodified counterpart in WT mouse liver. d Quantitation of the MATα1 Ser114 in normal (n = 4) and AH (n = 5) human livers (p = 0.03). The center line, bounds of box, and whiskers represent mean, 25th to 75th percentile range, and minimum to maximum range. Normal liver is shown in blue and AH in red. e IP analysis of AML-12 and f HepG2 overexpressing MATα1 WT or S114A cell lysates using anti-His-Tag antibody followed by western blot analysis against PIN1. *p < 0.05. Statistical significance was determined by using two-tailed, one-sample t-test for treatment comparisons and ANOVA test for group comparisons. Results are shown as mean ± SEM. Source data are provided as a Source data file. AH alcoholic hepatitis, EV empty vector, IP immunoprecipitation, pSer phosphor-serine, WT wild type.

Article Snippet: For PIN1 knockdown 50 nM siRNA against human PIN1 (SR303529, Origene) or mouse Pin1 (SR403748, Origene) or equivalent scramble control (SC) were delivered into HepG2 or AML-12 cells by Lipofectamine RNAiMAX (Life Technologies) for 48 h following the manufacturer’s protocol.

Techniques: Phospho-proteomics, Binding Assay, Western Blot, Control, Recombinant, Quantitation Assay, Two Tailed Test, Plasmid Preparation, Immunoprecipitation

Journal: Nature communications

Article Title: Depletion of mitochondrial methionine adenosyltransferase α1 triggers mitochondrial dysfunction in alcohol-associated liver disease.

doi: 10.1038/s41467-022-28201-2

Figure Lengend Snippet: Fig. 5 Blocking the interaction with PIN1 protects MATα1 against alcohol-induced downregulation. a MAT enzymatic activity measured as methionine consumption and SAMe production using recombinant MATα1 WT and S114A proteins. b MAT1A mRNA levels (n = 3 independent experiments, p = 0.007 for WT ethanol vs control and p = 0.0002 S114A ethanol vs control), c western blot, and d corresponding densitometry analysis of AML-12 cells lysates after transfection with EV, MATα1 WT or S114A vectors and ethanol treatment using anti-His-Tag antibody (n = 3 independent experiments, p = 0.003 for WT ethanol vs control and p = 0.005 S114A ethanol vs WT ethanol). e Western blot analysis of AML-12 cells expressing MATα1 WT or S114A and treated with ethanol before CHX addition and graph on the right showing the t1/2 for MATα1 WT and S114A with and without ethanol treatment. f Western blot and g densitometry analysis of the mitochondrial fraction of AML-12 cells expressing MATα1 WT or S114A after ethanol treatment (n = 3 independent experiments, p = 0.003 for WT ethanol vs control and p = 0.016 S114A ethanol vs WT ethanol). h His-Tag (red) and TOM70 (green) immunofluorescence and mitochondrial. *p < 0.05. Statistical significance was determined by using two-tailed, one-sample t-test for treatment comparisons and ANOVA test for group comparisons. Results are shown as mean ± SEM. Source data are provided as a Source data file. WT is shown in blue and S114A is shown in red. CHX cycloheximide, EV empty vector, WT wild type.

Article Snippet: For PIN1 knockdown 50 nM siRNA against human PIN1 (SR303529, Origene) or mouse Pin1 (SR403748, Origene) or equivalent scramble control (SC) were delivered into HepG2 or AML-12 cells by Lipofectamine RNAiMAX (Life Technologies) for 48 h following the manufacturer’s protocol.

Techniques: Blocking Assay, Activity Assay, Recombinant, Control, Western Blot, Transfection, Expressing, Two Tailed Test, Plasmid Preparation

Journal: Nature communications

Article Title: Depletion of mitochondrial methionine adenosyltransferase α1 triggers mitochondrial dysfunction in alcohol-associated liver disease.

doi: 10.1038/s41467-022-28201-2

Figure Lengend Snippet: Fig. 6 Blocking PIN1-MATα1 interaction protects against alcohol-induced mitochondrial injury by increasing MATα1 mitochondrial content. a Mitotracker staining (red) and b quantification (n = 10 pictures/3 independent experiments, p = 0.002 for WT ethanol vs control; p = 0.047 for S114A ethanol vs control; p = 0.013 S114A ethanol vs WT ethanol), c mitochondrial membrane potential (n = 3 independent experiments, p = 0.045 for WT ethanol vs control; p = 0.022 S114A ethanol vs WT ethanol), d mitochondrial respiration and basal respiration (p = 0.015 for WT ethanol vs control; p = 0.049 S114A ethanol vs control; p = 0.035 WT ethanol vs S114A ethanol), ATP production (p = 0.022 for WT ethanol vs control; p = 0.041 S114A ethanol vs control; p = 0.041 WT ethanol vs S114A ethanol) and maximal respiratory capacity (p = 0.005 for WT ethanol vs control; p = 0.040 S114A ethanol vs control; p = 0.004 WT ethanol vs S114A ethanol from 3 independent experiments), e ATP (n = 3 independent experiments, p = 0.035 for WT ethanol vs control; p = 0.022 S114A ethanol vs control; p = 0.041 WT ethanol vs S114A ethanol), f mROS levels (n = 3 independent experiments, p = 0.013 for WT ethanol vs control; p = 0.04 WT ethanol vs S114A ethanol), g triglycerides content (n = 3 independent experiments, p = 0.017 for WT ethanol vs control; p = 0.008 WT ethanol vs S114A ethanol), and h Oil red O staining in AML-12 cells expressing MATα1 WT or S114A after ethanol treatment. *p < 0.05. Statistical significance was determined by using two-tailed, one-sample t-test for treatment comparisons and ANOVA test for group comparisons. Results are shown as mean ± SEM. Source data are provided as a Source data file. WT is shown in blue and S114A is shown in red. AA antimycin A, FCCP Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, mROS mitochondrial reactive oxygen species, OCR oxygen consumption rate, Oligom oligomycin, Rot rotenone, WT wild type.

Article Snippet: For PIN1 knockdown 50 nM siRNA against human PIN1 (SR303529, Origene) or mouse Pin1 (SR403748, Origene) or equivalent scramble control (SC) were delivered into HepG2 or AML-12 cells by Lipofectamine RNAiMAX (Life Technologies) for 48 h following the manufacturer’s protocol.

Techniques: Blocking Assay, Staining, Control, Membrane, Expressing, Two Tailed Test

Journal: Nature communications

Article Title: Depletion of mitochondrial methionine adenosyltransferase α1 triggers mitochondrial dysfunction in alcohol-associated liver disease.

doi: 10.1038/s41467-022-28201-2

Figure Lengend Snippet: Fig. 9 PIN1 impairs MATα1 mitochondrial targeting iEn alcohol- associated liver disease. Graphical summary of the study. Alcohol promotes CK2 phosphorylation of MATα1 at Ser114 and interaction with PIN1, thereby inhibiting MATα1 mitochondrial targeting. Alcohol-induced mitochondrial MATα1 depletion contributes to mitochondrial dysfunction and the pathogenesis of ALD.

Article Snippet: For PIN1 knockdown 50 nM siRNA against human PIN1 (SR303529, Origene) or mouse Pin1 (SR403748, Origene) or equivalent scramble control (SC) were delivered into HepG2 or AML-12 cells by Lipofectamine RNAiMAX (Life Technologies) for 48 h following the manufacturer’s protocol.

Techniques: Phospho-proteomics

Journal: Nucleic Acids Research

Article Title: The fission yeast Pin1 peptidyl-prolyl isomerase promotes dissociation of Sty1 MAPK from RNA polymerase II and recruits Ssu72 phosphatase to facilitate oxidative stress induced transcription

doi: 10.1093/nar/gkaa1243

Figure Lengend Snippet: S. pombe strains used in this study

Article Snippet: Anti-Pin1 antibody was from R&D Systems (MAB2294, MN, USA).

Techniques:

Journal: Nucleic Acids Research

Article Title: The fission yeast Pin1 peptidyl-prolyl isomerase promotes dissociation of Sty1 MAPK from RNA polymerase II and recruits Ssu72 phosphatase to facilitate oxidative stress induced transcription

doi: 10.1093/nar/gkaa1243

Figure Lengend Snippet: Pin1 mutant is defective in oxidative stress induced transcription. ( A ) Tenfold serial dilution of strains indicated were spotted onto YES-agar plates without (control) or with 2 mM H 2 O 2 and photographed after 3 days incubation at 30°C. ( B ) H 2 O 2 (2 mM) was added to an asynchronous culture of strains indicated growing at 30°C in YES medium. Cell viability was measured at the time indicated by plating appropriate dilution of cells onto YES agar plate without drug and scoring colony formation after 3 days incubation at 30°C. Viability (% survival) is expressed as a percentage of the number of colonies obtained before the addition of drug. ( C ) Expression of stress-responsive genes indicated upon oxidative stress in wild type, pin1 Δ, atf1 Δ and pin1 Δ atf1 Δ cells. Total RNA from cultures of the indicated strains grown at 30°C was reverse transcribed using an oligo(dT) primer. The cDNA was amplified using quantitative PCR and SYBR green with primers specific for the indicated genes. The amount of RNA normalized to cdc2 mRNA relative to the wild type was expressed as means ± SD in triplicate. Asterisks indicated statistical significance ( P < 0.05, t -test unpaired) verse wild type.

Article Snippet: Anti-Pin1 antibody was from R&D Systems (MAB2294, MN, USA).

Techniques: Mutagenesis, Serial Dilution, Control, Incubation, Expressing, Reverse Transcription, Amplification, Real-time Polymerase Chain Reaction, SYBR Green Assay

Journal: Nucleic Acids Research

Article Title: The fission yeast Pin1 peptidyl-prolyl isomerase promotes dissociation of Sty1 MAPK from RNA polymerase II and recruits Ssu72 phosphatase to facilitate oxidative stress induced transcription

doi: 10.1093/nar/gkaa1243

Figure Lengend Snippet: Pin1 mutant is not defective in MAPK-mediated signal transduction pathway. ( A ) Whole-cell protein extracts of the strains indicated were prepared by alkaline extraction followed by trichloroacetic acid precipitation. The extracts were separated by SDS-PAGE and subjected to Western immunoblotting using anti-HA antibodies to reveal proteins of interest. Anti p-p38 antibody cross-reacted with phosphorylated Sty1. Antibody against α-tubulin was used as the loading control. ( B ) ChIP assays showing recruitment of Atf1-myc to the promoter of ctt1 , gpd1 , hsp9 and cdc2 genes upon oxdative stress. The latter was not induced upon stress. Samples were prepared from atf1-myc and atf1-myc pin1 Δ cells treated with 2 mM H 2 O 2 for the time points indicated. The DNA recovered from the IP was assayed by PCR using primers specific to the promoter of genes indicated. The control lanes show DNA amplified from whole cell extracts (WCE) prior to performing the IP. ( C ) ChIP assays showing recruitment of Atf1-myc and Sty1-myc to the promoter of ctt1 , gpd1 and hsp9 genes upon oxdative stress in pin1 Δ mutant and wild type cells. Samples were prepared from strains indicated treated with 2 mM H 2 O 2 for the time points indicated. The DNA recovered from the IP was amplified using quantitative PCR and SYBR green with primers specific to the promoter of genes indicated normalized to that of the non-stress responsive cdc2 gene. Data were expressed as means ± SD in triplicate. Asterisks indicated statistical significance ( P < 0.05, t -test unpaired) verse wild type.

Article Snippet: Anti-Pin1 antibody was from R&D Systems (MAB2294, MN, USA).

Techniques: Mutagenesis, Transduction, Extraction, TCA Precipitation, SDS Page, Western Blot, Control, Amplification, Real-time Polymerase Chain Reaction, SYBR Green Assay

Journal: Nucleic Acids Research

Article Title: The fission yeast Pin1 peptidyl-prolyl isomerase promotes dissociation of Sty1 MAPK from RNA polymerase II and recruits Ssu72 phosphatase to facilitate oxidative stress induced transcription

doi: 10.1093/nar/gkaa1243

Figure Lengend Snippet: Pin1 mutant is defective in the oxidative stress induced transcription response at the initiation to elongation transition. ( A ) Whole-cell protein extracts of the strains indicated were prepared by alkaline extraction followed by trichloroacetic acid precipitation. The extracts were separated by SDS-PAGE analyzed phosphorylation by western immunoblotting with CTD phospho-specific antibodies. Antibody against α-tubulin was used as the control. The relative level of phosphorylated protein was shown in ( B ). Data were expressed as means±SD in triplicate. Asterisks indicated statistical significance ( P <0.05, t-test unpaired) verse wild type. ( C ) ChIP assays showing the recruitment of Rpb1 to the promoter and the gene coding regions (ORF) of ctt1 , gpd1 and hsp9 genes upon oxidative stress in wild type cells and pin1 Δ mutant. Samples were prepared from the indicated strains treated with 2 mM H 2 O 2 for the time points indicated. The DNA recovered from the IP was amplified using quantitative PCR and SYBR green with primers specific to the promoter of the indicated genes normalized to that of the non-stress responsive cdc2 gene. Data were expressed as means ± SD in triplicate. Asterisks indicated statistical significance ( P < 0.05, t -test unpaired) verse wild type. ( D ) ChIP assays showing the recruitment of pSer5-Rpb1 to the promoters of ctt1 , gpd1 and hsp9 genes upon oxdative stress in wild type cells and pin1 Δ mutant as in ( C ). Data were expressed as means ± SD in triplicate. Asterisks indicated statistical significance ( P < 0.05, t -test unpaired) verse wild type.

Article Snippet: Anti-Pin1 antibody was from R&D Systems (MAB2294, MN, USA).

Techniques: Mutagenesis, Extraction, TCA Precipitation, SDS Page, Phospho-proteomics, Western Blot, Control, Amplification, Real-time Polymerase Chain Reaction, SYBR Green Assay

Journal: Nucleic Acids Research

Article Title: The fission yeast Pin1 peptidyl-prolyl isomerase promotes dissociation of Sty1 MAPK from RNA polymerase II and recruits Ssu72 phosphatase to facilitate oxidative stress induced transcription

doi: 10.1093/nar/gkaa1243

Figure Lengend Snippet: Sty1 interacted and phosphorylated Rpb1-CTD at Ser5. ( A ) Coimmunoprecipitation was performed with extracts prepared from indicated strains treated with 2 mM H 2 O 2 for 30 min. GFP-Trap affinity resin was used to pull down GFP proteins. Immunoprecipitates were analyzed by Western immunoblotting using antibodies against GFP and Rpb1-CTD. The relative level of Rpb1 pull down by Sty1 and its phosphorylation status was shown in ( B ). Data were expressed as means±SD in triplicate. Asterisks indicated statistical significance ( P < 0.05, t -test unpaired) verse wild type. ( C ) Coimmunoprecipitation was performed with extracts prepared from Pin1-GFP tagged strains treated with 2 mM H 2 O 2 for 30 min. GFP-Trap affinity resin was used to pull down GFP proteins. Immunoprecipitates were treated with/without CIP and analyzed by Western immunoblotting using antibodies against GFP and Rpb1-CTD. *Degradation product of Pin1-GFP. The relative level of Rpb1 pull down by Pin1 was shown. Data were expressed as means±SD in triplicate. Asterisks indicated statistical significance ( P <0.05, t-test unpaired) verse control of no treatments. ( D , E ) Purified GST-CTD from Escherichia coli and recombinant protein of Sty1 from yeast cells were subjected to GST pulldown assay followed by Western immunoblotting analysis with antibodies against GST and His. ( F ) In vitro kinase assay using Sty1-His extracted and immunoprecipitated from activated cells. Following incubation with GST-CTD, proteins were resolved by SDS-PAGE and probed with antibodies indicated.

Article Snippet: Anti-Pin1 antibody was from R&D Systems (MAB2294, MN, USA).

Techniques: Western Blot, Phospho-proteomics, Control, Purification, Recombinant, GST Pulldown Assay, In Vitro, Kinase Assay, Immunoprecipitation, Incubation, SDS Page

Journal: Nucleic Acids Research

Article Title: The fission yeast Pin1 peptidyl-prolyl isomerase promotes dissociation of Sty1 MAPK from RNA polymerase II and recruits Ssu72 phosphatase to facilitate oxidative stress induced transcription

doi: 10.1093/nar/gkaa1243

Figure Lengend Snippet: A mutual exclusive binding mode of Rpb1 to Pin1 over Sty1. ( A ) Recombinant proteins of Sty1 and CTD-GFP from yeast cells were subjected to GFP pulldown assay in the presence or absence of Pin1 followed by Western immunoblotting analysis with antibodies indicated. *Degradation product of Pin1-GST. The relative level of Sty1 pull down by CTD-GFP was shown. Data were expressed as means±SD in triplicate. Asterisks indicated statistical significance ( P <0.05, t-test unpaired) verse control without Pin1. ( B ) Whole-cell protein extracts of the strains indicated were prepared by alkaline extraction followed by trichloroacetic acid precipitation. The extracts were separated by SDS-PAGE and subjected to immunoblotting using anti-His antibodies to reveal Pin1 proteins. Antibody against α-tubulin was used as the control. ( C ) Tenfold serial dilutions of strains indicated were spotted onto EMM+thiamine agar plates without (control) or with 2 mM H 2 O 2 and photographed after 3 days incubation at 30°C. ( D ) Coimmunoprecipitation was performed with extracts prepared from Sty1-GFP tagged strains treated with 0.5 M NaCl for the time point indicated. GFP-Trap affinity resin was used to pull down GFP proteins. Immunoprecipitates were analyzed by Western immunoblotting using antibodies against GFP and Rpb1-CTD. The relative level of Rpb1 pull down by Sty1 was shown. Data were expressed as means±SD in triplicate. Asterisks indicated statistical significance ( P < 0.05, t-test unpaired) verse control of no treatment. ( E ) Tenfold serial dilutions of strains indicated were spotted onto YES-agar plates without (control) or with 200 mM NaCl and photographed after 3 days incubation at 30°C.

Article Snippet: Anti-Pin1 antibody was from R&D Systems (MAB2294, MN, USA).

Techniques: Binding Assay, Recombinant, Western Blot, Control, Extraction, TCA Precipitation, SDS Page, Incubation

Journal: Nucleic Acids Research

Article Title: The fission yeast Pin1 peptidyl-prolyl isomerase promotes dissociation of Sty1 MAPK from RNA polymerase II and recruits Ssu72 phosphatase to facilitate oxidative stress induced transcription

doi: 10.1093/nar/gkaa1243

Figure Lengend Snippet: Pin1 directly interacts and recruits Ssu72 for pSer5 dephosphorylation. ( A ) Whole-cell protein extracts of the strains indicated were prepared by alkaline extraction followed with trichloroacetic acid precipitation. The extracts were separated by SDS-PAGE and analyzed phosphorylation by Western immunoblotting with CTD phospho-specific antibodies. Antibody against α-tubulin was used as the control. The relative level of phosphorylated protein was shown in ( B ). Data were expressed as means±SD in triplicate. Asterisks indicated statistical significance ( P < 0.05, t -test unpaired) verse wild type. ( C ) In vitro phosphatase activity assay using recombinant protein of CTD-GFP from yeast cells were treated with Ssu72 recombinant protein or 20 units calf intestinal alkaline phosphatase (CIP) at 30°C for 1 h. Reaction mixtures were resolved by SDS-PAGE and probed with antibodies indicated. ( D ) Coimmunoprecipitation was performed with extracts prepared from Ssu72-myc tagged strains expressing Pin1-GFP and being treated with 2 mM H 2 O 2 for 30 min. GFP-Trap affinity resin was used to pull down GFP proteins. Immunoprecipitates were analyzed by western immunoblotting using antibodies against GFP and myc. *Degradation product of Pin1-GFP. The relative level of Ssu72 pull down by Pin1-GFP was shown. Data were expressed as means±SD in triplicate. ( E ) Recombinant proteins of Pin1-GST and Ssu72-His were subjected to GST pull down assay followed by Western immunoblotting analysis with antibodies against GST and His. *Degradation product of Pin1-GST. ( F ) Recombinant proteins of Ssu72-His and Pin1-His were subjected to GFP pulldown assay by CTD-GFP followed by Western immunoblotting analysis with antibodies against GFP and His. ( G ) ChIP assays showing the recruitment of Pin1-myc and Ssu72-myc to the promoters of ctt1 , gpd1 and hsp9 genes upon oxdative stress. Samples were prepared from strains indicated treated with 2 mM H 2 O 2 for the time points indicated. The DNA recovered from the IP was amplified using quantitative PCR and SYBR green with primers specific to the promoters of genes indicated normalized to that of the non-stress responsive cdc2 gene. Data was expressed as means ± SD in triplicate. Asterisks indicated statistical significance ( P < 0.05, t -test unpaired) verse control of no treatment. ( H ) Strains indicated were subjected to measurement of intracellular ROS levels in triplicate using CM-H 2 DCFDA as indicator. Asterisks indicated statistical significance ( P < 0.05, t -test unpaired) verse wild type. ( I ) Tenfold serial dilutions of strains indicated were spotted onto YES-agar plates without (control) or with 2 mM H 2 O 2 and photographed after 3 days incubation at 30°C. Growth characteristics of the strains indicated at 30°C (doubling time) were shown on the right. Data were expressed as means ± SD in triplicate. Asterisks indicated statistical significance ( P < 0.05, t -test unpaired) verse wild type.

Article Snippet: Anti-Pin1 antibody was from R&D Systems (MAB2294, MN, USA).

Techniques: De-Phosphorylation Assay, Extraction, TCA Precipitation, SDS Page, Phospho-proteomics, Western Blot, Control, In Vitro, Phosphatase Assay, Recombinant, Expressing, Pull Down Assay, Amplification, Real-time Polymerase Chain Reaction, SYBR Green Assay, Incubation

Journal: Nucleic Acids Research

Article Title: The fission yeast Pin1 peptidyl-prolyl isomerase promotes dissociation of Sty1 MAPK from RNA polymerase II and recruits Ssu72 phosphatase to facilitate oxidative stress induced transcription

doi: 10.1093/nar/gkaa1243

Figure Lengend Snippet: The characteristic feature of the fission yeast Pin1 identified is conserved in humans. ( A ) Coimmunoprecipitation was performed with extracts prepared from human HeLa cells expressing p38 tagged with flag. Immunoprecipitates were analyzed by Western immunoblotting analysis using antibodies specific for Rpb1 and flag. The relative level of Rpb1 pull down by p38 was shown. Data were expressed as means±SD in triplicate. Asterisks indicated statistical significance ( P < 0.05, t -test unpaired) verse control of no treatment. ( B ) Purified GST-CTD from Escherichia coli and p38-flag precipitated from HeLa cells were subjected to GST pulldown assay followed by Western immunoblotting analysis with antibodies against GST and flag. ( C ) In vitro kinase assay using p38 extracted and immunoprecipitated from activated cells. Following incubation with GST-CTD, proteins were resolved by SDS-PAGE and probed with antibodies indicated. ( D ) Coimmunoprecipitation was performed with extracts prepared from human HeLa cells using anti-Ssu72 antibody and IgG as control. Immunoprecipitates were analyzed by Western immunoblotting analysis with antibodies specific for human Pin1 and Ssu72. ( E ) Total lysates of human cell indicated were subjected to Western immunoblotting analysis with indicated antibodies. The relative level of protein of interest was indicated beneath each lane. Data were expressed as means±SD in triplicate. Asterisks indicated statistical significance ( P < 0.05, t -test unpaired) verse RWPE-1 cells. ( F ) ( G ) Human cells indicated were subjected to measurement of intracellular ROS Levels in triplicate using CM-H 2 DCFDA as indicator. Data were expressed as means±SD in triplicate. Asterisks indicated statistical significance ( P < 0.05, t -test unpaired) verse normal RWPE-1 cells.

Article Snippet: Anti-Pin1 antibody was from R&D Systems (MAB2294, MN, USA).

Techniques: Expressing, Western Blot, Control, Purification, GST Pulldown Assay, In Vitro, Kinase Assay, Immunoprecipitation, Incubation, SDS Page

Journal: Nucleic Acids Research

Article Title: The fission yeast Pin1 peptidyl-prolyl isomerase promotes dissociation of Sty1 MAPK from RNA polymerase II and recruits Ssu72 phosphatase to facilitate oxidative stress induced transcription

doi: 10.1093/nar/gkaa1243

Figure Lengend Snippet: Model of Sty1-mediated oxidative stress induced transcription. Upon oxidative stress, activated Sty1 phosphorylates the CTD reiterated heptad sequence at Serine 5. Pin1 competes the binding of phosphorylated CTD to promote Sty1 dissociating from it, and directly recruits Ssu72 to facilitate dephosphorylation of CTD for transcription elongation.

Article Snippet: Anti-Pin1 antibody was from R&D Systems (MAB2294, MN, USA).

Techniques: Sequencing, Binding Assay, De-Phosphorylation Assay

Journal: Nucleic Acids Research

Article Title: The fission yeast Pin1 peptidyl-prolyl isomerase promotes dissociation of Sty1 MAPK from RNA polymerase II and recruits Ssu72 phosphatase to facilitate oxidative stress induced transcription

doi: 10.1093/nar/gkaa1243

Figure Lengend Snippet: Pin1 and Ssu72-dependent genes respond to oxidative stress

Article Snippet: Anti-Pin1 antibody was from R&D Systems (MAB2294, MN, USA).

Techniques: Binding Assay

Journal: Magnetochemistry (Basel, Switzerland)

Article Title: Efficient Stereospecific H β2/3 NMR Assignment Strategy for Mid-Size Proteins

doi: 10.3390/magnetochemistry4020025

Figure Lengend Snippet: Correlation between 3 J Hα–Hβ coupling values in Pin1 obtained from non-uniform and linear sampling. The error bars were calculated using .

Article Snippet: Human wild-type Pin1 was cloned into pET-28a(+) (Genscript) with an N-terminal 6xhistidine tag.

Techniques: Sampling

Journal: Magnetochemistry (Basel, Switzerland)

Article Title: Efficient Stereospecific H β2/3 NMR Assignment Strategy for Mid-Size Proteins

doi: 10.3390/magnetochemistry4020025

Figure Lengend Snippet: Number of stereospecific assignments determined using the presented pulse sequence paired with CYANA calculations.

Article Snippet: Human wild-type Pin1 was cloned into pET-28a(+) (Genscript) with an N-terminal 6xhistidine tag.

Techniques: Sequencing

Journal: Magnetochemistry (Basel, Switzerland)

Article Title: Efficient Stereospecific H β2/3 NMR Assignment Strategy for Mid-Size Proteins

doi: 10.3390/magnetochemistry4020025

Figure Lengend Snippet: Root-mean-square deviations (RMSDs) for various structure calculations for the ensemble compared with its average, as well as with the crystal structure (PDB code 1pin) [ 16 ].

Article Snippet: Human wild-type Pin1 was cloned into pET-28a(+) (Genscript) with an N-terminal 6xhistidine tag.

Techniques:

Journal: Magnetochemistry (Basel, Switzerland)

Article Title: Efficient Stereospecific H β2/3 NMR Assignment Strategy for Mid-Size Proteins

doi: 10.3390/magnetochemistry4020025

Figure Lengend Snippet: Correlation between 3 J Hα–Hβ coupling values in the isolated WW domain, and the WW domain of full-length Pin1. For the isolated WW domain, the originally published pulse sequence was used, whereas the full-length Pin1 was evaluated in this study. The two strongest outliers are highlighted.

Article Snippet: Human wild-type Pin1 was cloned into pET-28a(+) (Genscript) with an N-terminal 6xhistidine tag.

Techniques: Isolation, Sequencing

Journal: Molecular carcinogenesis

Article Title: Peptidyl-prolyl cis/trans isomerase Pin1 regulates withaferin A-mediated cell cycle arrest in human breast cancer cells

doi: 10.1002/mc.22814

Figure Lengend Snippet: Withaferin A (WA) treatment downregulated protein level of Pin1 in breast cancer in vivo and in vitro. (A) Structure of WA. (B) Representative immunohistochemical images showing Pin1 expression in 3 different tumor sections of control- and WA-treated MMTV-neu mice (200× magnification; scale bar = 20 μm). (C) Quantitation of Pin1 expression in breast tumor sections of MMTV-neu mice. The bar graph shows mean Pin1 expression (H-score, positive pixel algorithm) with standard deviation (n = 7 for both control and WA treatment groups). *Statistically significant (P < 0.05) compared with control group by unpaired Student’s t-test. (D) Western blot for Pin1 protein using breast tumor supernatants from control- and WA-treated rats. (E) Bar graph showing quantitation of Pin1 protein expression in rat tumors (mean ± SD; n = 6). (F) Representative confocal microscopic images (63× oil objective magnification; scale bar = 200 μm) showing Pin1 (green fluorescence) expression in MCF-7 and SK-BR-3 cells following 24 h treatment with DMSO or WA (2 μM). DRAQ5 (blue fluorescence) was used for nuclear staining. The results were consistent in replicate independent experiments.

Article Snippet: Recombinant human Pin1 protein was purchased from MyBioSource (San Diego, CA).

Techniques: In Vivo, In Vitro, Immunohistochemical staining, Expressing, Control, Quantitation Assay, Standard Deviation, Western Blot, Fluorescence, Staining

Journal: Molecular carcinogenesis

Article Title: Peptidyl-prolyl cis/trans isomerase Pin1 regulates withaferin A-mediated cell cycle arrest in human breast cancer cells

doi: 10.1002/mc.22814

Figure Lengend Snippet: Pin1 overexpression in MCF-7 and SK-BR-3 cells attenuated WA-mediated mitotic arrest (A) Western blotting for Pin1 protein using lysates from MCF-7 and SK-BR-3 cells treated for 24 h with DMSO (control) or WA. (B) Western blotting for Pin1 protein using lysates from pcDNA3 empty vector (1) and myc-tagged Pin1 (2) transfected cells. Western blotting was performed at least twice. (C) Representative flow histograms depicting mitotic fraction (indicated by an arrow) in pcDNA3 empty vector transfected MCF-7 cells and those transfected with myc-tagged Pin1 and treated for 24 h with DMSO or 1 μM of WA. (D) Quantitation of mitotic fraction in MCF-7 cells. Combined results from two experiments are shown as mean ± SD (n = 6). (E) Quantitation of G2/M phase cells. Combined results from three experiments are shown as mean ± SD (n = 9). (F) Quantitation of total [early (Annexin V-positive) and late (Annexin V-positive and propidium iodide-positive)] apoptosis in pcDNA3 empty vector transfected MCF-7 cells and those transfected with myc-tagged Pin1 and treated for 24 h or 48 h with DMSO or WA. Combined results from two experiments are shown as mean ± SD (n = 5~6). Effect of myc-Pin1 overexpression on WA-mediated (24 h treatment) (G) mitotic arrest and (H) total apoptosis in SK-BR-3 cells. Experiments were done at least twice with consistent results and representative data from one such experiment are shown as mean ± SD (n = 3). Statistically significant (P < 0.05) compared with the *corresponding DMSO-treated control or #between pcDNA3 and myc-Pin1 groups by one-way ANOVA followed by Bonferroni’s multiple comparison test.

Article Snippet: Recombinant human Pin1 protein was purchased from MyBioSource (San Diego, CA).

Techniques: Over Expression, Western Blot, Control, Plasmid Preparation, Transfection, Quantitation Assay, Comparison

Journal: Molecular carcinogenesis

Article Title: Peptidyl-prolyl cis/trans isomerase Pin1 regulates withaferin A-mediated cell cycle arrest in human breast cancer cells

doi: 10.1002/mc.22814

Figure Lengend Snippet: Withaferin A (WA) treatment modulated expression of cell cycle regulatory proteins in MCF-7 and SK-BR-3 cells. Western blotting for Cdc25C, Cdc2, Cyclin B1, and β-Tubulin using lysates from pcDNA3 empty vector transfected and myc-tagged Pin1 overexpressing MCF-7 and SK-BR-3 cells treated for 24 h with DMSO or indicated doses of WA. Experiments were done at least twice and results were consistent.

Article Snippet: Recombinant human Pin1 protein was purchased from MyBioSource (San Diego, CA).

Techniques: Expressing, Western Blot, Plasmid Preparation, Transfection

Journal: Molecular carcinogenesis

Article Title: Peptidyl-prolyl cis/trans isomerase Pin1 regulates withaferin A-mediated cell cycle arrest in human breast cancer cells

doi: 10.1002/mc.22814

Figure Lengend Snippet: Withaferin A (WA) interacted covalently with Cys113 of human Pin1. (A) Model of the Cys113-WA adduct of human Pin1. Right bottom, comprehensive view of the model. The Cys113-WA adduct is shown as a sphere model with an atomic color scheme (carbon in green, oxygen in red, and hydrogen in white). Left, particulars of the WA-binding pocket. The human Pin1 model is shown as a ribbon diagram (helices as spirals, strands as arrows, and loops as tubes) with WA-interacting side chains as sticks (nitrogen in blue, carbon in gray, and oxygen in red), and the Cys113-WA adduct is shown as a stick model (nitrogen in blue, carbon in green, oxygen in red, and sulfur in yellow). (B) Human Pin1 peptide sequence IKSGEEDFESLASQFSDCSSAK. (C) Pin1 peptide sequence showing b-ions and y-ions. (D) Tandem mass spectrum of peptide IKSGEEDFESLASQFSDCSSAK with sequence-specific product ions (b18 and y5) confirming the presence of the Cys113-WA adduct.

Article Snippet: Recombinant human Pin1 protein was purchased from MyBioSource (San Diego, CA).

Techniques: Binding Assay, Sequencing

Journal: Molecular carcinogenesis

Article Title: Peptidyl-prolyl cis/trans isomerase Pin1 regulates withaferin A-mediated cell cycle arrest in human breast cancer cells

doi: 10.1002/mc.22814

Figure Lengend Snippet: Pin1 overexpression augmented WA-mediated increase in expression of proapoptotic IGFBP family proteins. (A) Human antibody array map with proapoptotic proteins numbered. (B) Antibody array showing expression of apoptosis regulating proteins. The antibody array was performed using lysates from pcDNA3 empty vector transfected MCF-7 cells or those transfected with myc-tagged Pin1 after 24 h treatment with DMSO or 1 μM of WA. (C) Bar graph showing levels of proapoptotic proteins relative to DMSO-treated MCF-7 cells transfected with the empty vector (pcDNA3; mean ± SD; n = 2). Dotted lines indicate ± 2-fold changes in relative protein levels (i.e.≥2 or ≤ 0.5).

Article Snippet: Recombinant human Pin1 protein was purchased from MyBioSource (San Diego, CA).

Techniques: Over Expression, Expressing, Ab Array, Plasmid Preparation, Transfection

Journal: Molecular carcinogenesis

Article Title: Peptidyl-prolyl cis/trans isomerase Pin1 regulates withaferin A-mediated cell cycle arrest in human breast cancer cells

doi: 10.1002/mc.22814

Figure Lengend Snippet: Pin1 overexpression and WA treatment altered the expression of anti-apoptotic proteins in MCF-7 cells. (A) Human antibody array map with anti-apoptotic proteins numbered. (B) Antibody array showing expression of anti-apoptotic proteins. Antibody array was performed using lysates from pcDNA3 empty vector transfected MCF-7 cells or those overexpressing myc-tagged Pin1 after 24 h treatment with DMSO or 1 μM of WA. (C) Bar graph showing levels of anti-apoptotic proteins relative to DMSO-treated MCF-7 cells transfected with the empty vector (pcDNA3; mean ± SD; n = 2). Dotted lines indicate ± 2-fold changes in relative protein levels (i.e. ≥ 2 or ≤ 0.5).

Article Snippet: Recombinant human Pin1 protein was purchased from MyBioSource (San Diego, CA).

Techniques: Over Expression, Expressing, Ab Array, Plasmid Preparation, Transfection