Journal: bioRxiv

Article Title: Pin1-promoted SUMOylation of RNF168 restrains its chromatin accumulation

doi: 10.1101/2022.03.22.485326

Figure Lengend Snippet: A. Diagram depicting domain architecture of RNF168. The location of potential Pin1 binding motifs in RNF168 are highlighted with blue circles. B. U2OS cells were treated with siNTC and siPin1 for 72 hrs. Later cells were fixed and stained for RNF168 and γ-H2AX. Scale bars 10 μm.

Article Snippet: The following antibodies were used in this study: rabbit anti RNF168 polyclonal Ab (1:1000 dilution, Millipore, ABE367), mouse anti myc mAb (1:1000 dilution, CST, 2276), mouse anti Pin1 mAb (1:2000 dilution, R&D Systems, MAB2294), rabbit anti Histone H3 polyclonal Ab (1:2000 dilution, Abcam, ab1791), rabbit anti γ-H2AX polyclonal Ab (1:1000 dilution, Abcam, ab2893), rabbit anti H2AX polyclonal Ab (1:1000 dilution, Abcam, ab11175), mouse anti SUMO1 mAb (1:500 dilution, Millipore, MABS2071), mouse anti SUMO2/3 mAb (1:1000 dilution, Abcam, ab81371), rabbit anti RNF8 mAb (1:1000 dilution, Abcam, ab128872) mouse anti Tubulin mAb (1:1000 dilution, Santa Cruz, sc-5286), rabbit anti Vinculin mAb (1:2000 dilution, Abcam, ab129002), rabbit anti-mouse-HRP polyclonal Ab (1:5000 dilution, DAKO, P0161), Swine anti-rabbit polyclonal Ab (1:5000 dilution, DAKO, P0217).

Techniques: Binding Assay, Staining

Journal: bioRxiv

Article Title: Pin1-promoted SUMOylation of RNF168 restrains its chromatin accumulation

doi: 10.1101/2022.03.22.485326

Figure Lengend Snippet: A. Representative images of RNF168 foci after treatment with ionising radiation (IR). U2OS cells were transfected with non-targeting control (NTC) or Pin1 siRNA for 72 hrs and treated with IR, 2 Gy. 1 hr after IR cells were fixed and stained for endogenous RNF168 and γ-H2AX. Scale bars 10 μm. B. Quantification of RNF168 foci intensity from A. Data is mean ± s.e.m, n= 170 cells. p-value <0.0001. C. Western blot of Pin1 protein following treatment with non-targetting siRNA (siNTC) and Pin1 siRNA (siPin1). D. Representative images of exogenous RNF168 foci. U2OS cells stably expressing myc-RNF168 were treated with siNTC or siPin1 for 72 hrs and stained for myc and γ-H2AX. Scale bars 10 μm. The right panel shows the fluorescence intensity profiles along the line for myc and γ-H2AX. E. Quantification of myc-RNF168 foci intensity from D. Data is mean ± s.e.m, n= 42 cells for siNTC and 48 cells for siPin1. p-value <0.0001. F. Representative images of myc-RNF168 foci formation after IR. U2OS cells expressing myc-RNF168 were treated with indicated siRNAs and stained for myc and γ-H2AX after 1 hr post IR, 2 Gy. Scale bars 10 μm. G. Quantification of myc-RNF168 foci intensity from F. Data is mean ± s.e.m, n= 54 cells for siNTC and 66 cells for siPin1. p-value <0.0001. H. Western blot of chromatin fraction or whole cell lysate (WCL) for myc, histone H3, Pin1 and tubulin. U2OS cells expressing myc-RNF168 were treated with siNTC or siPin1 for 72 hrs. Cells were treated with 10 Gy of IR and collected after indicated time points to prepare chromatin fraction or WCL. SE: short exposure, LE: Long exposure.

Article Snippet: The following antibodies were used in this study: rabbit anti RNF168 polyclonal Ab (1:1000 dilution, Millipore, ABE367), mouse anti myc mAb (1:1000 dilution, CST, 2276), mouse anti Pin1 mAb (1:2000 dilution, R&D Systems, MAB2294), rabbit anti Histone H3 polyclonal Ab (1:2000 dilution, Abcam, ab1791), rabbit anti γ-H2AX polyclonal Ab (1:1000 dilution, Abcam, ab2893), rabbit anti H2AX polyclonal Ab (1:1000 dilution, Abcam, ab11175), mouse anti SUMO1 mAb (1:500 dilution, Millipore, MABS2071), mouse anti SUMO2/3 mAb (1:1000 dilution, Abcam, ab81371), rabbit anti RNF8 mAb (1:1000 dilution, Abcam, ab128872) mouse anti Tubulin mAb (1:1000 dilution, Santa Cruz, sc-5286), rabbit anti Vinculin mAb (1:2000 dilution, Abcam, ab129002), rabbit anti-mouse-HRP polyclonal Ab (1:5000 dilution, DAKO, P0161), Swine anti-rabbit polyclonal Ab (1:5000 dilution, DAKO, P0217).

Techniques: Transfection, Control, Staining, Western Blot, Stable Transfection, Expressing, Fluorescence

Journal: bioRxiv

Article Title: Pin1-promoted SUMOylation of RNF168 restrains its chromatin accumulation

doi: 10.1101/2022.03.22.485326

Figure Lengend Snippet: A. Pull down of endogenous RNF168 by GST-fused WW or W34A mutant domain of Pin1 from U2OS cells. B. Pull down of endogenous RNF168 by GST-fused WW or W34A mutant domain of Pin1 from HeLa cells. C. Pull down of myc-RNF168 by GST-fused WW or W34A mutant domain of Pin1 from U2OS cells treated with 10Gy of IR. Cell lysate were made at indicated time points after IR and subjected for WW domain pull-down. D. U2OS cells expressing myc-RNF168 wild type (WT) or T208A mutant were subjected to pull down by GST-fused-WW or W34A domain of Pin1. E. Western blot of chromatin fraction or WCL probed for myc, histone H3 and vinculin. U2OS cells were treated with siRNAs targeting RNF168 for 72 hrs to deplete endogenous RNF168. Cells were simultaneously treated with 1 μg/ml doxycycline to induce expression of siRNA resistant myc-RNF168-WT or T208A mutant. Chromatin fraction and WCL were prepared following treatment with 10 Gy of IR at indicated time points. F. Representative images of foci formation of exogenously expressed RNF168 mutants. U2OS cells were treated with 1 μg/ml doxycycline for 72 hrs to induce expression of siRNA resistant myc-RNF168-WT, T208A, P209A and T208A/P209A mutants. Endogenous RNF168 was depleted simultaneously by treatment with siRNF168. Cells were fixed and stained for myc and γ-H2AX. Scale bars 10 μm. G. As in F, cells expressing RNF168-WT, T208A, P209A, T208A/P209A mutants were treated with 2 Gy of IR and fixed after 1hr. Cells were stained for myc and γ-H2AX. Scale bars 10 μm. H. Quantification of myc-RNF168 mutant foci intensity from F. Data is mean ± s.e.m, n= 73 for WT, 75 for T208A, 92 for P209A and 73 for T208A/P209A. p-value <0.0001 (****). I. Quantification of myc-RNF168 mutant foci intensity from G. Data is mean ± s.e.m, n= 152 for WT, 142 for T208A, 130 for P209A and 163 for T208A/P209A. p-value <0.0001 (****).

Article Snippet: The following antibodies were used in this study: rabbit anti RNF168 polyclonal Ab (1:1000 dilution, Millipore, ABE367), mouse anti myc mAb (1:1000 dilution, CST, 2276), mouse anti Pin1 mAb (1:2000 dilution, R&D Systems, MAB2294), rabbit anti Histone H3 polyclonal Ab (1:2000 dilution, Abcam, ab1791), rabbit anti γ-H2AX polyclonal Ab (1:1000 dilution, Abcam, ab2893), rabbit anti H2AX polyclonal Ab (1:1000 dilution, Abcam, ab11175), mouse anti SUMO1 mAb (1:500 dilution, Millipore, MABS2071), mouse anti SUMO2/3 mAb (1:1000 dilution, Abcam, ab81371), rabbit anti RNF8 mAb (1:1000 dilution, Abcam, ab128872) mouse anti Tubulin mAb (1:1000 dilution, Santa Cruz, sc-5286), rabbit anti Vinculin mAb (1:2000 dilution, Abcam, ab129002), rabbit anti-mouse-HRP polyclonal Ab (1:5000 dilution, DAKO, P0161), Swine anti-rabbit polyclonal Ab (1:5000 dilution, DAKO, P0217).

Techniques: Mutagenesis, Expressing, Western Blot, Staining

Journal: bioRxiv

Article Title: Pin1-promoted SUMOylation of RNF168 restrains its chromatin accumulation

doi: 10.1101/2022.03.22.485326

Figure Lengend Snippet: A. U2OS cells expressing myc-RNF168-WT or S197A or T230A mutant were subjected to pull down by GST-fused-WW or W34A domain of Pin1. B. U2OS cells were treated siRNF168 for 72 hrs and 1μg/ml doxycycline to induce expression of myc-RNF168-WT, S197A, T208A, T230A. Endogenous RNF168 was depleted simultaneously by treatment with siRNF168. Cells were treated with 2 Gy of IR. 1hr after IR, cells were fixed and stained for myc and γ-H2AX. Scale bars 10 μm. C. Western blot of chromatin fraction and WCL for myc, histone H3 and vinculin. Cells complemented with RNF168-WT, S197A and T230A were treated with 10 Gy of IR. 1 hr later, untreated or IR treated cells were collected to prepare chromatin and WCL fractions. D. U2OS cells complemented with myc-RNF168-T208A were treated with 1 μg/ml doxycycline and siRNF8 for 72 hrs. Cells were treated with 2 Gy of IR and fixed 1 hr later. Cells were stained for myc and γ-H2AX. Scale bars 10 μm. E. Western blot to show depletion of RNF8 for D.

Article Snippet: The following antibodies were used in this study: rabbit anti RNF168 polyclonal Ab (1:1000 dilution, Millipore, ABE367), mouse anti myc mAb (1:1000 dilution, CST, 2276), mouse anti Pin1 mAb (1:2000 dilution, R&D Systems, MAB2294), rabbit anti Histone H3 polyclonal Ab (1:2000 dilution, Abcam, ab1791), rabbit anti γ-H2AX polyclonal Ab (1:1000 dilution, Abcam, ab2893), rabbit anti H2AX polyclonal Ab (1:1000 dilution, Abcam, ab11175), mouse anti SUMO1 mAb (1:500 dilution, Millipore, MABS2071), mouse anti SUMO2/3 mAb (1:1000 dilution, Abcam, ab81371), rabbit anti RNF8 mAb (1:1000 dilution, Abcam, ab128872) mouse anti Tubulin mAb (1:1000 dilution, Santa Cruz, sc-5286), rabbit anti Vinculin mAb (1:2000 dilution, Abcam, ab129002), rabbit anti-mouse-HRP polyclonal Ab (1:5000 dilution, DAKO, P0161), Swine anti-rabbit polyclonal Ab (1:5000 dilution, DAKO, P0217).

Techniques: Expressing, Mutagenesis, Staining, Western Blot

Journal: bioRxiv

Article Title: Pin1-promoted SUMOylation of RNF168 restrains its chromatin accumulation

doi: 10.1101/2022.03.22.485326

Figure Lengend Snippet: A. HEK293 cells containing shRNA control (NTC) or shPin1 were transfected with myc-RNF168 alone or in combination with 6xHis-Flag-SUMO2. Cells were treated with 1 mM IPTG for 72 hrs for Pin1 depletion. 10 μM MG132 was added for 6 hrs before lysate preparation. SUMO2 conjugated proteins were enriched by Ni 2+ beads under denaturing conditions. * = non-specific band. B. HEK293 cells complemented with various RNF168 variants were transfected with 6xHis-Flag-SUMO2. Cells were treated with 1 μg/ml doxycycline for 72 hrs. Later SUMO conjugated proteins were pulled down with Ni 2+ beads under denaturing conditions. SUMO conjugated RNF168 variants were detected by western blotting. * = non-specific band. C. Colony survival of U2OS cells depleted of RNF168 and complemented with WT, T208A, P209A and T208A/P209A variants of myc-RNF168 after treatment with indicated doses of IR. The number of replicates n for each condition is mentioned in parentheses. Data is mean ± s.e.m. D. Western blot to show depletion of RNF168 and complementation of RNF168 variants for C. E. Colony survival of U2OS cells depleted of RNF168 and complemented with WT, T208A, K210R and P209A/K210R variants of myc-RNF168 after treatment with indicated doses of IR. The number of replicates n for each condition is mentioned in parentheses. Data is mean ± s.e.m. F. Western blot to show depletion of RNF168 and complementation of RNF168 variants for E.

Article Snippet: The following antibodies were used in this study: rabbit anti RNF168 polyclonal Ab (1:1000 dilution, Millipore, ABE367), mouse anti myc mAb (1:1000 dilution, CST, 2276), mouse anti Pin1 mAb (1:2000 dilution, R&D Systems, MAB2294), rabbit anti Histone H3 polyclonal Ab (1:2000 dilution, Abcam, ab1791), rabbit anti γ-H2AX polyclonal Ab (1:1000 dilution, Abcam, ab2893), rabbit anti H2AX polyclonal Ab (1:1000 dilution, Abcam, ab11175), mouse anti SUMO1 mAb (1:500 dilution, Millipore, MABS2071), mouse anti SUMO2/3 mAb (1:1000 dilution, Abcam, ab81371), rabbit anti RNF8 mAb (1:1000 dilution, Abcam, ab128872) mouse anti Tubulin mAb (1:1000 dilution, Santa Cruz, sc-5286), rabbit anti Vinculin mAb (1:2000 dilution, Abcam, ab129002), rabbit anti-mouse-HRP polyclonal Ab (1:5000 dilution, DAKO, P0161), Swine anti-rabbit polyclonal Ab (1:5000 dilution, DAKO, P0217).

Techniques: shRNA, Control, Transfection, Western Blot

Journal: International Journal of Biological Sciences

Article Title: S-Adenosylmethionine Negatively Regulates the Mitochondrial Respiratory Chain Repressor MCJ in the Liver

doi: 10.7150/ijbs.90104

Figure Lengend Snippet: MCJ protein levels are induced in the livers of Mat1a -KO mice . (A) MATα1 and MCJ protein levels by western blotting and densitometric quantification in liver lysates from WT and Mat1a -KO mice. (B) Dnajc15 expression measured by RT-PCR in livers from WT and Mat1a -KO mice. (C) MCJ protein levels by western blotting and densitometric quantification in primary hepatocytes (PH) from WT and Mat1a -KO mice. (D) MCJ protein levels by western blotting and densitometric quantification in cytosolic and mitochondrial fractions from WT and Mat1a -KO mouse livers. *P<0.05 Mat1a -KO vs WT.

Article Snippet: For MAT1A knockdown 50 nM siRNA against human MAT1A (SR303529, Origene) or mouse Mat1a (161996, Thermo Scientific) or equivalent scramble control (SC) were delivered into mouse hepatocytes or HepG2 cells by Lipofectamine RNAiMAX (Life Technologies) for 48 hours following the manufacturer's protocol.

Techniques: Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: International Journal of Biological Sciences

Article Title: S-Adenosylmethionine Negatively Regulates the Mitochondrial Respiratory Chain Repressor MCJ in the Liver

doi: 10.7150/ijbs.90104

Figure Lengend Snippet: MAT1A negatively regulates MCJ via SAMe . (A) MCJ and MATα1 protein levels by western blotting and densitometric quantification and (B) Dnajc15 and Mat1a expression measured by RT-PCR in mouse primary hepatocytes (PH) after Mat1a silencing. (C) MCJ and MATα1 protein levels by western blotting and densitometric quantification and (D) DNAJC15 and MAT1A expression measured by RT-PCR in HepG2 cells after MAT1A silencing. (E) MCJ and MATα1 levels by western blotting and densitometric quantification (lower panel) after MAT1A overexpression in SAMe-D cells. (F-H) MCJ and MATα1 levels by western blotting in Huh-7 cells after (F) MAT1A WT and (G) the catalytic mutant overexpression and (H) densitometric quantification. (I) DNAJC15 and MAT1A expression measured by RT-PCR in Huh-7 cells after MAT1A WT and catalytic mutant overexpression. *P<0.05 si MAT1A vs SC; MAT1A OV vs EV; MAT1A mutant OV vs EV.

Article Snippet: For MAT1A knockdown 50 nM siRNA against human MAT1A (SR303529, Origene) or mouse Mat1a (161996, Thermo Scientific) or equivalent scramble control (SC) were delivered into mouse hepatocytes or HepG2 cells by Lipofectamine RNAiMAX (Life Technologies) for 48 hours following the manufacturer's protocol.

Techniques: Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Over Expression, Mutagenesis

Journal: International Journal of Biological Sciences

Article Title: S-Adenosylmethionine Negatively Regulates the Mitochondrial Respiratory Chain Repressor MCJ in the Liver

doi: 10.7150/ijbs.90104

Figure Lengend Snippet: SAMe reduces MCJ protein levels in hepatocytes. (A) MCJ protein levels by western blotting, (B) quantification and (C) DNAJC15 mRNA levels in human primary hepatocytes (PH), mouse PH, HepG2, Huh-7 and SAMe-D cells after 24 hours of SAMe treatment. (D) MCJ-HA levels measured by western blotting in Huh-7 cells after MCJ overexpression for 48 hours and 24 hours of SAMe treatment. (E) MCJ and MATα1 levels by western blotting and densitometric quantification in WT, Mat1a -KO and Mat1a -KO PH treated with SAMe for 24 hours. (F) MCJ and MATα1 protein levels by western blotting and densitometric quantification in mouse PHs after isolation (0h) and cultured for 24 hours with or without SAMe. *P<0.05 SAMe vs Control; Mat1a -KO vs WT; 24 hours vs 0 hours and # P<0.05 Mat1a -KO + SAMe vs Mat1a -KO; 24 hours + SAMe vs 24 hours.

Article Snippet: For MAT1A knockdown 50 nM siRNA against human MAT1A (SR303529, Origene) or mouse Mat1a (161996, Thermo Scientific) or equivalent scramble control (SC) were delivered into mouse hepatocytes or HepG2 cells by Lipofectamine RNAiMAX (Life Technologies) for 48 hours following the manufacturer's protocol.

Techniques: Western Blot, Over Expression, Isolation, Cell Culture, Control

Journal: International Journal of Biological Sciences

Article Title: S-Adenosylmethionine Negatively Regulates the Mitochondrial Respiratory Chain Repressor MCJ in the Liver

doi: 10.7150/ijbs.90104

Figure Lengend Snippet: MATα1 and MCJ interact in liver mitochondria. (A) Immunoprecipitation (IP) analyses using MATα1 antibody in mouse liver lysates and (B) human hepatocyte lysates. (C) IP analyses using MCJ (left panel) and MATα1 antibodies (right panel) in mouse liver mitochondrial lysates. (D) IP analyses using MATα1 antibody in human liver mitochondrial lysates. (E) IP analysis using MATα1 and MCJ recombinant proteins and beads conjugated to anti- MATα1 and anti-MCJ antibodies. IgG was used as a negative control. (F) IP analyses in mouse liver lysates using antibodies against methyl-lysine and methyl-arginine. (G) Extracted ion chromatograms (XIC) for a doubly charged peptide indicative of MCJ K83 di-methylation. ISSPSFSSYYKGGFEQK has a co-eluting precursor trace with site specific product ions for MCJ K83 di-methylation and is differentially expressed between WT and Mat1a -KO. (H-J) MCJ levels by western blotting (upper panels) and quantification (lower panels) in (H) Huh-7, (I) HepG2 cells and (J) mouse PHs treated with SAMe for 24 hours before cycloheximide (CHX) addition. Graphs show MCJ's t 1/2 with and without SAMe treatment. *P<0.05 SAMe vs Control.

Article Snippet: For MAT1A knockdown 50 nM siRNA against human MAT1A (SR303529, Origene) or mouse Mat1a (161996, Thermo Scientific) or equivalent scramble control (SC) were delivered into mouse hepatocytes or HepG2 cells by Lipofectamine RNAiMAX (Life Technologies) for 48 hours following the manufacturer's protocol.

Techniques: Immunoprecipitation, Recombinant, Negative Control, Methylation, Western Blot, Control

Journal: International Journal of Biological Sciences

Article Title: S-Adenosylmethionine Negatively Regulates the Mitochondrial Respiratory Chain Repressor MCJ in the Liver

doi: 10.7150/ijbs.90104

Figure Lengend Snippet: SAMe does not regulate MCJ via proteasomal or autophagosomal degradation. (A) MCJ and MATα1 levels by western blotting and quantification in HepG2 cells after MAT1A silencing for 48 hours and treatment with the proteasome inhibitor MG132 for the last 6 hours. (B) MCJ and MATα1 levels by western blotting and quantification in PH from WT and Mat1a -KO mice treated with the proteasome inhibitor MG132 for 6 hours. (C) MCJ, MATα1 and protein ubiquitination levels by western blotting and quantification (table below) in mouse PH, HepG2, and Huh-7 cells after treatment with SAMe for 24 hours and the proteasome inhibitor MG132 (20μM) for the last 6 hours. (D) MCJ, MATα1 and LC3 levels by western blotting and quantification (table below) in mouse PH, HepG2, and Huh-7 cells after treatment with SAMe for 24 hours and the autophagy inhibitor chloroquine (CQ) (50μM) for the last 6 hours. *P<0.05 MG132 vs Control; SAMe vs Control; MG132 or CQ vs Control; SAMe+MG132 or SAMe+CQ vs Control; # P<0.05 si MAT1A vs SC; Mat1a -KO vs WT; MG132 or CQ vs SAMe; SAMe+MG132 or SAMe+CQ vs SAMe. † P<0.05 SAMe+MG132 or SAMe+CQ vs MG132 or CQ.

Article Snippet: For MAT1A knockdown 50 nM siRNA against human MAT1A (SR303529, Origene) or mouse Mat1a (161996, Thermo Scientific) or equivalent scramble control (SC) were delivered into mouse hepatocytes or HepG2 cells by Lipofectamine RNAiMAX (Life Technologies) for 48 hours following the manufacturer's protocol.

Techniques: Western Blot, Ubiquitin Proteomics, Control

Journal: International Journal of Biological Sciences

Article Title: S-Adenosylmethionine Negatively Regulates the Mitochondrial Respiratory Chain Repressor MCJ in the Liver

doi: 10.7150/ijbs.90104

Figure Lengend Snippet: MCJ contributes to mitochondrial dysfunction caused by MAT1A deficiency. (A) MCJ and MATα1 levels by western blotting and densitometric quantification, (B) DNAJC15 and MAT1A mRNA levels by RT-PCR, (C) ATP levels, (D) mitochondrial ROS (mROS) production, (E) mitochondrial respiration (left panel) and basal respiration, ATP production and maximal respiratory capacity (right panel) using the Seahorse analyzer in HepG2 cells after MAT1A, DNAJC15 or MAT1A+DNAJC15 silencing for 48 hours. *P<0.01 si MAT1A vs SC; si MAT1A +si DNAJC15 vs si DNAJC15. # P<0.01 si DNAJC15 vs SC; si MAT1A +si DNAJC15 vs SC . † P<0.05 si MAT1A +si DNAJC15 vs si MAT1A. (F) MCJ and MATα1 levels by western blotting and densitometric quantification, (G) Dnajc15 and Mat1a mRNA levels by RT-PCR, (H) ATP levels, (I) mitochondrial ROS (mROS) production, (J) mitochondrial respiration (left panel) and basal respiration, ATP production and maximal respiratory capacity (right panel) using the Seahorse analyzer in primary hepatocytes (PH) from WT and Mat1a -KO mice cells after Dnajc15 silencing for 36 hours. *P<0.01; si Dnajc15 vs SC. # P<0.01 Mat1a -KO SC vs WT SC. † P<0.05 Mat1a -KO si Dnajc15 vs WT si Dnajc15.

Article Snippet: For MAT1A knockdown 50 nM siRNA against human MAT1A (SR303529, Origene) or mouse Mat1a (161996, Thermo Scientific) or equivalent scramble control (SC) were delivered into mouse hepatocytes or HepG2 cells by Lipofectamine RNAiMAX (Life Technologies) for 48 hours following the manufacturer's protocol.

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction

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: 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: 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