Journal: PLoS ONE

Article Title: Differential Impact of Tetratricopeptide Repeat Proteins on the Steroid Hormone Receptors

doi: 10.1371/journal.pone.0011717

Figure Lengend Snippet: HEK-293 cells were transfected as described for , except that HA-MR was expressed instead of HA-GR. Cells were processed and protein interactions were analyzed also as described for . In A, binding of TPR-proteins is presented relative to the mean of the normalized FLAG-eluate signals of CHIP, FKBP51, and TPR2. Quantification represents means of three independent experiments (two for XAP2) +S.E.M. In B, binding is normalized as in . C, FLAG- and HA-immunoblot signals of the cell extracts, demonstrating expression of TPR proteins and AR. Quantifications represent means of three independent experiments +S.E.M.

Article Snippet: Non-specific binding to membrane was blocked by 5% nonfat milk in Tris-buffered saline supplemented with 0.1% Tween-20, and then one of the following specific primary antibodies were added: Actin (I-19, Santa-Cruz), FLAG tag-HRP (M2, Sigma); hemagglutinin tag-HRP (Roche Applied Science); p23 (ABR), FKBP52 (Anti-FKBP59, Stressgen), CHIP (PC711, Calbiochem), Cyp40 (ABR, PA3-022), FKBP51 (F14, Santa Cruz), XAP2 (ARA9, NB100-127, Novus Biologicals), TPR2 (kind gift of Ulrich Hartl), PP5/PPT (BD Biosciences).

Techniques: Transfection, Binding Assay, Western Blot, Expressing

Journal:

Article Title: Structural basis for controlling the dimerization and stability of the WW domains of an atypical subfamily

doi: 10.1110/ps.035329.108

Figure Lengend Snippet: (A) The neighbor-joining (NJ) tree dendrogram of the WW domains from human origin. The WW domain sequences from human origin were obtained from the Pfam server (http://www.sanger.ac.uk/Software/Pfam/), and the redundancy was removed using the 100% sequence identity filter. The sequences thus selected were analyzed using the program CLUSTAL W (Thompson et al. 1994). (Magenta) The members of the subclass that contains SAV1 WW2 and MAGI1 WW2. (B) Comparative representation of the LOGO plot of the representative WW domain sequences and the sequences of the members of the subclass that contains SAV1 WW2 and MAG1 WW2. (Yellow boxes) Polar residues of E242 and S246 in SAV1 WW2, as well as those of their counterparts in MAGI1 WW2, E366 and D370. (Red) Acidic residues, (blue) basic residues.

Article Snippet: The DNA encoding the human MAGI1 gene was subcloned by PCR from the human cDNA clone locus NM_004742 (TrueClone Collection, OriGene, Cat No. SC117150).

Techniques: Software, Sequencing

Journal:

Article Title: Structural basis for controlling the dimerization and stability of the WW domains of an atypical subfamily

doi: 10.1110/ps.035329.108

Figure Lengend Snippet: Equilibrium analytical ultracentrifugation of MAGI1 WW2 355–390. (Bottom panel) Radial distribution of the absorbance in the centrifuge cell at equilibrium at 16,000 rpm, with a protein concentration of 0.5 mg/mL. The solid line through the data represents the fit to a single species with a molecular weight corresponding to 5611, while the theoretical molecular weight for the single polypeptide chain is 5580. (Upper panels) Residuals for the fit.

Article Snippet: The DNA encoding the human MAGI1 gene was subcloned by PCR from the human cDNA clone locus NM_004742 (TrueClone Collection, OriGene, Cat No. SC117150).

Techniques: Analytical Ultracentrifugation, Protein Concentration, Molecular Weight

Journal:

Article Title: Structural basis for controlling the dimerization and stability of the WW domains of an atypical subfamily

doi: 10.1110/ps.035329.108

Figure Lengend Snippet: (A) The 1H-15N HSQC spectrum of MAGI1 WW2 355–390, with labels. (*) Signals from the N- and C-terminal artificial tags of the present construct. (B) Solution structures of MAGI1 WW2 355–390, in line representations. (Cyan) Residues in the N- and C-terminal artificial tags of the present construct.

Article Snippet: The DNA encoding the human MAGI1 gene was subcloned by PCR from the human cDNA clone locus NM_004742 (TrueClone Collection, OriGene, Cat No. SC117150).

Techniques: Construct

Journal:

Article Title: Structural basis for controlling the dimerization and stability of the WW domains of an atypical subfamily

doi: 10.1110/ps.035329.108

Figure Lengend Snippet: Structural comparison between mSAV1 WW2 231–266 and hMAGI1 WW2 355–390. (A) Structure of SAV1 WW2 231–266. (Lines) Side chains of key aromatic residues (F249, Y252, H256, Y263) buried in the interface of the homodimer; (neon bars) side chains of the polar residues (E242, S246). These side chains are colored to clarify the representation of the molecular interaction. (B) Structure of MAGI1 WW2 355–390. (Lines) Side chains of the aromatic residues (Y373, Y376, H380, Y387); (neon bars) side chains of the polar residues (E366, D370).

Article Snippet: The DNA encoding the human MAGI1 gene was subcloned by PCR from the human cDNA clone locus NM_004742 (TrueClone Collection, OriGene, Cat No. SC117150).

Techniques: Comparison

Journal:

Article Title: Structural basis for controlling the dimerization and stability of the WW domains of an atypical subfamily

doi: 10.1110/ps.035329.108

Figure Lengend Snippet: (A) The 1H-15N HSQC spectrum of MAGI1 WW2 355–401, with labels. (*) Signals from the N- and C-terminal artificial tags of the present construct. (B) Solution structures of MAGI1 WW2 355–401, in line representations. (Cyan) Residues in the N- and C-terminal artificial tags of the present construct.

Article Snippet: The DNA encoding the human MAGI1 gene was subcloned by PCR from the human cDNA clone locus NM_004742 (TrueClone Collection, OriGene, Cat No. SC117150).

Techniques: Construct

Journal:

Article Title: Structural basis for controlling the dimerization and stability of the WW domains of an atypical subfamily

doi: 10.1110/ps.035329.108

Figure Lengend Snippet: The 15N spin relaxation profiles of MAGI1 WW2 355–390 (black), MAGI1 WW2 355–401 (red), and MAGI1 WW2 355–390 D370S (green). The heteronuclear NOE (top), longitudinal (R1; middle), and transverse (R2; bottom) relaxation profiles are shown with error bars. (Cyan arrows) β-strands, (red bar) the α-helix of the longer construct, MAGI1 WW2 355–401.

Article Snippet: The DNA encoding the human MAGI1 gene was subcloned by PCR from the human cDNA clone locus NM_004742 (TrueClone Collection, OriGene, Cat No. SC117150).

Techniques: Construct

Journal:

Article Title: Structural basis for controlling the dimerization and stability of the WW domains of an atypical subfamily

doi: 10.1110/ps.035329.108

Figure Lengend Snippet: Thermal stability summary of MAGI1 WW2 355–390 and its variants

Article Snippet: The DNA encoding the human MAGI1 gene was subcloned by PCR from the human cDNA clone locus NM_004742 (TrueClone Collection, OriGene, Cat No. SC117150).

Techniques:

Journal:

Article Title: Structural basis for controlling the dimerization and stability of the WW domains of an atypical subfamily

doi: 10.1110/ps.035329.108

Figure Lengend Snippet: (A) Far-UV CD spectra of MAGI1 WW2 355–390 (black), MAGI1 WW2 355–401 (red), and MAGI1 WW2 355–390 D370S (green), recorded at 20°C. (B) Thermal denaturation profiles of MAGI1 WW2 355–390 (black), MAGI1 WW2 355–401 (red), and those of the D370S mutant, recorded with 55 μM (green) and 5 μM (blue) protein concentrations. The changes in ellipticity at 232 nm for MAGI1 WW2 355–390 and MAGI1 WW2 355–390 D370S, and that at 217 nm for MAGI1 WW2 355–401, were monitored and analyzed as a function of temperature.

Article Snippet: The DNA encoding the human MAGI1 gene was subcloned by PCR from the human cDNA clone locus NM_004742 (TrueClone Collection, OriGene, Cat No. SC117150).

Techniques: Circular Dichroism, Mutagenesis

Journal:

Article Title: Structural basis for controlling the dimerization and stability of the WW domains of an atypical subfamily

doi: 10.1110/ps.035329.108

Figure Lengend Snippet: (A) Close-up view highlighting the interactions around the C-terminal α-helix in MAGI1 WW2 355–401. Key residues described in the main text are marked. (B) Structural comparison between MAGI1 WW2 355–390 (blue) and MAGI1 WW2 355–401 (red). Structures were superimposed using the backbone heavy atoms in the region between Leu361 and Pro390. (C) Comparison of the accessible surface areas of the residues between MAGI1 WW2 355–390 (black) and MAGI1 WW2 355–401 (red). (Cyan bars) β-strands; (red bar) the α-helix of the longer construct, MAGI1 WW2 355–401.

Article Snippet: The DNA encoding the human MAGI1 gene was subcloned by PCR from the human cDNA clone locus NM_004742 (TrueClone Collection, OriGene, Cat No. SC117150).

Techniques: Comparison, Construct

Journal: Cell Biology and Toxicology

Article Title: SP1 and p23 play a crucial role in the circadian target gene induction of activated aryl hydrocarbon receptor in human breast cells

doi: 10.1007/s10565-025-10080-0

Figure Lengend Snippet: Circadian pattern in protein levels of the AHR cofactor SP1. a ) To identify AHR co-factors with a circadian expression level, the mRNA fold change of non-synchronized and synchronized HME1 cells after a treatment with 0.5 nM TCDD was analyzed at the indicated timepoints after synchronization. The mRNA levels of AHR , ARNT , XAP2 , SP1 , HSP90 , P23 and AHRR were analyzed by RT-qPCR. For each timepoint the fold change of the CYP1A1 expression was calculated based on the DMSO control of the respective timepoint. The TCDD mediated induction of AHR , ARNT , XAP2 , SP1 , HSP90 , and P23 remained stable over time in both non-synchronized and synchronized cells. The induction of the AHR target gene AHRR displayed under synchronized conditions a circadian pattern with peaking after 24 h and 48 h. Each bar represents the mean ± SD of three independent experiments. The individual data points from the single experiments are displayed. b) Representative Western blots from three independent experiments of the AHR co-factors AHR, ARNT, XAP2, SP1, HSP90 and p23 from HME1 cells after 12, 24, 36, and 48 h of synchronization. The protein levels of SP1 show a circadian pattern with peaking at 24 h and 48 h upon synchronization indicating that SP1 protein levels under circadian regulation. β-Actin and total protein stain served as loading control. The relative amounts of proteins were derived from the representative Western blot signal intensity and normalized to β-Actin. The quantification was conducted with Image Studio™ Lite Software (LI-COR Biosciences)

Article Snippet: The following primary antibodies diluted in 3% w/v BSA/TBST were used: AHR antibody (1:1000; Abcam; ab190797), ARNT antibody (1:1000; Cell Signaling; #3414), p23 antibody (1:10000; Invitrogen; #MA3-414), HSP90 antibody (1:1000; Cell Signaling; #4877), XAP2 antibody (1:1000; Novus Biologicals; NB100-127), SP1 antibody (1:2000; Novus Biologicals; NB600-233), CLOCK antibody (1:1000; Cell Signaling; #5157), BMAL1 antibody (1:1000; Novus Biologicals; NB100-2288), BMAL1 antibody (1:500; Santa Cruz; sc-365645), β-Actin antibody (1:5000, Abcam, ab8227) and GAPDH antibody (1:1000, Cell signaling; #5174).

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Staining, Derivative Assay, Software

Journal: Cell Biology and Toxicology

Article Title: SP1 and p23 play a crucial role in the circadian target gene induction of activated aryl hydrocarbon receptor in human breast cells

doi: 10.1007/s10565-025-10080-0

Figure Lengend Snippet: Modulator of the circadian regulated AHR activity. a ) Representative fitted curve of the circadian rhythm of M13SV1 cells transiently transfected with a PER2:LUCIFERASE (orange) or BMAL1:LUCIFERASE (blue) circadian reporter plasmid. M13SV1 were transfected via electroporation with the indicated reporter plasmids. 24 h after transfection confluent cells were synchronized by treatment with 1 µM dexamethasone for 1 h and subsequently monitored for 48h. The bioluminescence signal was recorded every 30 min and the obtained measurements was analyzed by the ChronAlyzer software. M13SV1 shows a stable synchronization with an anticyclic expression of the PER2 or BMAL1 control luciferase b) CYP1A1 induction at different time points in synchronized and non-synchronized M13SV1 cells upon treatment with 0.5 nM TCDD. CYP1A1 mRNA after TCDD exposure was analyzed by RT-qPCR at indicated timepoints. Fold change was calculated based on the DMSO control of the respective timepoint. In synchronized cells, the TCDD-mediated CYP1A1 induction displays a circadian pattern peaking at 24 h. Each bar represents the mean ± SD of three independent experiments. The individual data points from the single experiments are displayed. Statistical significance was determined by using an unpaired, one-tailed t-test. c) To determine the knock down efficiency of the targeted genes, the mRNA levels of the indicated genes of interest (GOI) were analyzed by RT-qPCR. The knockdown level was calculated by comparing the GOI with the control siRNA transfected cells. Each bar represents the mean ± SD of three independent experiments. The individual data points from the single experiments are displayed. d) Decrease and increase of CYP1A1 induction was determined in M13SV1 cells transfected with siRNA targeting the AHR or its co-factors ARNT , XAP2 , HSP90 and p23 . The transfected cells were synchronized with 1 µM dexamethasone or left non-synchronized and subsequently exposed to 0.5 nM TCDD for 24 h. CYP1A1 mRNA levels were determined by RT-qPCR and the change in induction was calculated for the siRNA-targeted gene compared to the control siRNA. Each bar represents the mean ± SD of three independent experiments. The individual data points from the single experiments are displayed.

Article Snippet: The following primary antibodies diluted in 3% w/v BSA/TBST were used: AHR antibody (1:1000; Abcam; ab190797), ARNT antibody (1:1000; Cell Signaling; #3414), p23 antibody (1:10000; Invitrogen; #MA3-414), HSP90 antibody (1:1000; Cell Signaling; #4877), XAP2 antibody (1:1000; Novus Biologicals; NB100-127), SP1 antibody (1:2000; Novus Biologicals; NB600-233), CLOCK antibody (1:1000; Cell Signaling; #5157), BMAL1 antibody (1:1000; Novus Biologicals; NB100-2288), BMAL1 antibody (1:500; Santa Cruz; sc-365645), β-Actin antibody (1:5000, Abcam, ab8227) and GAPDH antibody (1:1000, Cell signaling; #5174).

Techniques: Activity Assay, Transfection, Luciferase, Plasmid Preparation, Electroporation, Software, Expressing, Control, Quantitative RT-PCR, One-tailed Test, Knockdown