Journal: Acta Crystallographica Section F Structural Biology Communications

Article Title: Structure of human collapsin response mediator protein 1: a possible role of its C-terminal tail

doi: 10.1107/s2053230x15009243

Figure Lengend Snippet: Figure 1 Crystallization and SDS–PAGE analysis of purified human CRMP-1. (a) Morphology of CRMP-1 crystals. Left, the crystals after crystallization (2 d, 293 K). Right, the crystals after incubation (20 d). (b) Analysis (SDS–PAGE) of CRMP-1 proteins that had not (lane 1) or had (lane 2) been incubated with thrombin protease and a sample taken from a crystallization well containing high-quality CRMP-1 crystals after incubation (20 d at 293 K; lane 3). Samples (3 mg) were characterized using a Tris–glycine gel (12%) and stained with Coomassie Blue. (c) SDS– PAGE analysis of the purified C-terminal tail (residues 472–572) of CRMP-1. Samples were separated with a Tris–tricine (20%) peptide- separation gel and were detected with silver staining.

Article Snippet: Oligomerization analyses were performed with full-length and thrombin-cleaved CRMP-1 (using an analytical Superdex 200 10/300 column from GE Healthcare connected to an ÄKTA purifier system).

Techniques: Crystallization Assay, SDS Page, Incubation, Staining, Silver Staining

Journal: Acta Crystallographica Section F Structural Biology Communications

Article Title: Structure of human collapsin response mediator protein 1: a possible role of its C-terminal tail

doi: 10.1107/s2053230x15009243

Figure Lengend Snippet: Figure 2 A representative view of the final 2Fo Fc electron-density map (blue, resolution 3 A˚ , contoured at the 1.0 level), highlighting the fit of residues Arg467–Pro475 to the maps. The nonconsensus amino acid (depicted in red) in this region is Ala473 of human CRMP-1 (phenylalanine in mouse CRMP-1).

Article Snippet: Oligomerization analyses were performed with full-length and thrombin-cleaved CRMP-1 (using an analytical Superdex 200 10/300 column from GE Healthcare connected to an ÄKTA purifier system).

Techniques:

Journal: Acta Crystallographica Section F Structural Biology Communications

Article Title: Structure of human collapsin response mediator protein 1: a possible role of its C-terminal tail

doi: 10.1107/s2053230x15009243

Figure Lengend Snippet: Figure 4 Residues that are potentially important in determining interfaces in CRMP. The crystal structures of mouse CRMP-1 (yellow; PDB entry 1kcx; Deo et al., 2004), CRMP-2 (green; PDB entry 2gse; Stenmark et al., 2007), CRMP-4 (cyan; PDB entry 4bkn; Structural Genomics Consor- tium, unpublished work) and CRMP-5 (blue; PDB entry 4b91; Ponnusamy & Lohkamp, 2013) are superimposed onto human CRMP-1 (orange; PDB entry 4b3z). Both interfaces I (a) and II (b) contain conserved and nonconserved residues. The interacting residues are labelled for human CRMP-1; the corresponding residues of mouse CRMP-1, CRMP-2, CRMP-4 and CRMP-5 are given in parentheses.

Article Snippet: Oligomerization analyses were performed with full-length and thrombin-cleaved CRMP-1 (using an analytical Superdex 200 10/300 column from GE Healthcare connected to an ÄKTA purifier system).

Techniques:

Journal: Acta Crystallographica Section F Structural Biology Communications

Article Title: Structure of human collapsin response mediator protein 1: a possible role of its C-terminal tail

doi: 10.1107/s2053230x15009243

Figure Lengend Snippet: Figure 6 Oligomerization and characterization of the secondary structure of full- length and thrombin-cleaved CRMP-1. (a) Analytical gel-filtration chromatography of full-length (black line) and thrombin-cleaved (red line) CRMP-1 was performed as described in the Materials and methods; the profiles are superimposed based on the ‘inject’ signal. The elution volumes corresponding to the molecular masses of the protein markers is marked with blue arrows for comparison. Vo and Vt denote the void and total volumes of the column, respectively. Under these operating conditions, the apparent predominant species of full-length and thrombin-cleaved CRMP-1 are tetramers. (b) CD spectra of CRMP-1 (black line) and thrombin-cleaved CRMP-1 (red line) were recorded in the 260–200 nm wavelength region. In the inset in (b), CD spectra of the C-terminal tail of CRMP-1 (amino acids 472–572; grey line), the -helix- rich protein myoglobin (blue line) and the -strand-rich protein concanavalin A (green line) (each protein was dissolved in 10 mM Tris–HCl pH 7.4, 150 mMNaF) are shown in mean residue ellipticity units; the buffer baseline was subtracted.

Article Snippet: Oligomerization analyses were performed with full-length and thrombin-cleaved CRMP-1 (using an analytical Superdex 200 10/300 column from GE Healthcare connected to an ÄKTA purifier system).

Techniques: Chromatography, Comparison, Circular Dichroism, Residue

Journal: Molecular cell

Article Title: Native Chromatin Proteomics Reveals Role for Specific Nucleoporins in Heterochromatin Organization and Maintenance

doi: 10.1016/j.molcel.2019.10.018

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For each ChIP, approximately 2 μg of antibody was pre-incubated with 30 μl of Invitrogen Dynabeads Protein A, Protein G or IgG (for TAP tagged strains): anti-H3K9me2 (Abcam 1220) with protein A, anti-H3K14ac (Millipore 07–353) with protein-A, anti-Flag (Sigma F1804) with protein G, anti-Swi6 (rabbit polyclonal) with protein A, anti-GFP (Abcam 290) with protein A, and anti-Myc (Covance 9E10, MMS-150P) with protein G. For H3K9me3 ChIP, 1 μg of anti-H3K9me3 antibody (Diagenode, C15500003) was first incubated with 30 μl Dynabeads M-280 Streptavidin beads (Invitrogen) at 4 °C for 1 hr, followed by blocking with 5 μM biotin, according to the manufacturer’s instructions, prior to using for each immunoprecipitation.

Techniques: Purification, Recombinant, Silver Staining, Protease Inhibitor, Hybridization, Western Blot, Staining, Microscopy, Mass Spectrometry, Software, Real-time Polymerase Chain Reaction

Journal: Cell

Article Title: The Parkinson’s disease protein alpha-synuclein is a modulator of Processing-bodies and mRNA stability

doi: 10.1016/j.cell.2022.05.008

Figure Lengend Snippet: Key resources table

Article Snippet: BCA Protein Assay Kit , Pierce , 23225.

Techniques: Western Blot, Virus, Recombinant, Protease Inhibitor, Lysis, Magnetic Beads, Membrane, Transfection, Expressing, Bicinchoninic Acid Protein Assay, Silver Staining, In Situ, Sample Prep, Luciferase, Reporter Assay, Multiplex sample analysis, Biomarker Discovery, Marker, Generated, Software, Mass Spectrometry, Imaging

Journal:

Article Title: Saccharomyces cerevisiae DNA Polymerase ? and Polymerase ? Interact Physically and Functionally, Suggesting a Role for Polymerase ? in Sister Chromatid Cohesion

doi: 10.1128/MCB.23.8.2733-2748.2003

Figure Lengend Snippet: (A) Pol2 coimmunoprecipitates with Trf4. Coexpression of FLAG-Pol2 and His-Trf4 and immunoprecipitation protocols are described in Materials and Methods. Western blots of crude extract (labeled “I” for input protein) from insect cells expressing various combinations of Trf proteins and FLAG-Pol2 were probed with antibody against Trf4 or anti-FLAG Pol2 as indicated. Lane 1, no recombinant protein; lane 2, His-Trf4; lane 3, His-Trf4 plus FLAG-Pol2. These extracts were incubated with anti-FLAG beads. After washing of the beads, proteins that bound from extracts (labeled “B” for bound protein) were eluted by boiling. The proteins were analyzed on Western blots probed with antibody against Trf4 or anti-FLAG Pol2 as indicated on the right. Lane 4, no recombinant protein; lane 5, His-Trf4; lane 6, His-Trf4 plus FLAG-Pol2. (B) Pol2 coimmunoprecipitates with Trf5. Coexpression of FLAG-Pol2 and Trf5-His is described in Materials and Methods. Western blots of crude extract from insect cells are represented in the same order as those in Fig. ​Fig.2A.2A. (C) Recombinant Trf4 prepared in E. coli stimulates Pol ɛ holoenzyme. Trf4 was purified exactly as described previously (70). The oligo(dT)12-18 primer extension assay is described in Materials and Methods. Reaction mixtures contained 680 ng of Trf4, the amount required to observe Trf4 DNA polymerase activity (lanes 2 and 3), and/or 0.15 U of Pol ɛ, as indicated, are shown. The high level of Trf4 is saturating for stimulatory activity (see panel D). Lane 1, no protein; lane 2, Trf4-His with 0.1 mM dTTP; lane 3, Trf4-His with 1 mM dTTP; lane 4, Pol ɛ with 0.1 mM dTTP; lane 5, Pol ɛ with 1 mM dTTP; lane 6, Pol ɛ plus Trf4-His with 0.1 mM dTTP; and lane 7, Pol ɛ plus Trf4-His with 1 mM dTTP. (D) Titration of stimulatory activity of scTrf4 made in bacteria. The indicated amounts of scTrf4 were assayed for stimulation of [3H]dTMP incorporation by 0.15 U of Pol ɛ on an oligo(dT)-poly(dA) substrate as described in Materials and Methods. (E) scTrf4-His expressed in insect cell cochromatographs with Pol ɛ-stimulatory activity. scTrf4-His was expressed in insect cells. Silver staining of Trf4-His after purification through Ni2+-nitrilotriacetic acid and Mono Q columns, as described in Materials and Methods, and gel electrophoresis is shown at the top. Numbers refer to MonoQ fraction numbers. The same fractions are assayed for stimulation of primer extension by pol ɛ. Each fraction from the Mono Q column was dialyzed, and 2 μl of each fraction was used in a 20-μl reaction. Fraction 13 contained 17 ng of Trf4 protein (13 nM); but 2.7 nM Trf4 gave equivalent stimulation (not shown). The first lane shows no primer extension, the second lane shows activity of 0.15 U of Pol ɛ (0.5 nM) alone, and the subsequent lanes are the Mono Q fractions of the Trf4 purification assayed with 0.15 U of Pol ɛ (0.5 nm). The fraction numbers are identified above. (F) scTrf4-His and Pol ɛ-stimulatory activity copurify. [3H]dTMP incorporation assay: the same fractions from the Mono Q column were assayed for [3H]dTMP incorporation on an oligo(dT)-poly(dA) substrate as described in Materials and Methods in the presence of 0.15 U of Pol ɛ (0.5 nm). (G) Trf4 from the MonoQ column is highly purified. Coomassie-stained gel of Trf4 from fraction 14 of the MonoQ column. (H) scTrf4-His does not efficiently stimulate Pol2-140 lacking the C-terminal 1,000 amino acids. Three levels (0.075, 0.15, or 0.3 U) of either Pol ɛ (solid dots) or truncated Pol2 protein (open dots) were assayed with saturating amounts (40 ng) of Trf4-His purified from insect cells. Similar results were obtained with scTrf4 prepared in E. coli.

Article Snippet: As controls that the stimulation was not due to contaminating E. coli DNA polymerase I (the most abundant DNA polymerase in the bacterial extracts) in the scTrf4 preparation, we showed that DNA polymerase I of E. coli (Gibco BRL) did not stimulate Pol ɛ and antibody to E. coli Pol I (gift of S. Linn, U.C.

Techniques: Immunoprecipitation, Western Blot, Labeling, Expressing, Recombinant, Incubation, Purification, Primer Extension Assay, Activity Assay, Titration, Silver Staining, Nucleic Acid Electrophoresis, Staining

Journal: bioRxiv

Article Title: Molecular insights into the stimulation of SNM1A nuclease activity by CSB during interstrand crosslink processing

doi: 10.1101/2024.09.05.611390

Figure Lengend Snippet: A. N-Avi-Biotin-1′N-SNM1A (42 kDa) was immobilised on Streptavidin mutein matrix beads, washed with 0.5 M NaCl, then incubated with CSB protein: CSB 1187-1401 (26 kDa, appears 32 kDa), CSB 1308-1493 (23 kDa, appears 26 kDa) or CSB 1424-1493 (11 kDa). Unbound protein was then removed, the beads washed, and protein eluted. Samples were separated by SDS-PAGE (4-12% Bis-Tris). then analysed by silver staining. Lanes 0: Protein standards in kDa, 1: CSB protein alone, 2: CSB protein unbound to SNM1A, 3: wash 10/10 with 0.5 M NaCl, 4: elution. B . Apparent equilibrium dissociation (K D ) constants obtained for CSB domains for interaction with N-Avi-Biotin-SNM1A. The dark green region of the protein sequence map represents the amino acid ranges used for experiments. C. Single cycle response curves from SPR analyses, with apparent equilibrium dissociation constants. Parameters determined from fitting are given in Supplementary Table 4.

Article Snippet: N-Biotin-Avi-SNM1A (200 nM, 250 μL) was prepared in binding buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 50 mM KCl, 0.5 mM TCEP, 0.05% v/v Triton X-100, 5 mM MgCl 2 , 5% v/v glycerol, 1 mM phenylmethylsulfonyl chloride, 0.05% v/v NP-40) and immobilised on PierceTM Streptavidin magnetic beads (10 μL, ThermoFisher Scientific) by incubation at 4 °C for 1 h. The beads were then washed with 3 x 1 mL binding buffer with centrifugation at 1,000 x g , 4 °C, 2 min between each wash.

Techniques: Incubation, SDS Page, Silver Staining, Sequencing

Journal: bioRxiv

Article Title: Molecular insights into the stimulation of SNM1A nuclease activity by CSB during interstrand crosslink processing

doi: 10.1101/2024.09.05.611390

Figure Lengend Snippet: A. N-Avi-Biotin-1′N-SNM1A (42 kDa) was immobilised on Streptavidin mutein matrix beads, washed with 0.5 M NaCl, then incubated with CSB 1424-1493 (WT or T1447A, 11 kDa). Unbound protein was removed, the beads washed, and protein eluted. Samples were separated by SDS-PAGE (4-12% Bis-Tris) then analyzed by silver staining. Lanes 0: Protein standards in kDa, 1: CSB protein alone, 2: CSB protein unbound to SNM1A, 3: wash 10/10 with 0.5 M NaCl, 4: elution. B. SPR (dose response and multi-cycle analysis) with immobilized N-Avi-Biotin-SNM1A with WT-CSB 1424-1493 as the analyte. Response units are given. C. SPR (dose response and multi-cycle analysis) with immobilized N-Avi-Biotin-SNM1A with T1447A-CSB 1424-1493 as the analyte. Response units are given. D. N-Avi-Biotin-1′N-SNM1A (WT or E864A, appears 42 kDa) was immobilised on Streptavidin mutein matrix beads, then incubated with CSB 1424-1493 (WT or T1447A, 11 kDa). Unbound protein was removed, the beads washed, and protein eluted. Samples were separated by SDS-PAGE (4-12% Bis-Tris) then analysed by silver staining. Lanes 0: Protein standards in kDa, 1: CSB protein alone, 2: CSB protein unbound to SNM1A, 3: wash 10/10 with 0.5 M NaCl, 4: elution. E. Single cycle SPR spectroscopy with N-Avi-Biotin-1′N-SNM1A (WT or E864A) immobilised on the chip and CSB 1424-1493 or CSB 1187-1493 as the analyte. Dissociation constants with other parameters are given in Supplementary Table 4. F. Single cycle SPR spectroscopy with N-His-ZB-CSB-Avi-Biotin immobilized on the chip with untagged SNM1A (WT or E864A) as the analyte. Dissociation constants are given with other parameters determined in Supplementary Table 5.

Article Snippet: N-Biotin-Avi-SNM1A (200 nM, 250 μL) was prepared in binding buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 50 mM KCl, 0.5 mM TCEP, 0.05% v/v Triton X-100, 5 mM MgCl 2 , 5% v/v glycerol, 1 mM phenylmethylsulfonyl chloride, 0.05% v/v NP-40) and immobilised on PierceTM Streptavidin magnetic beads (10 μL, ThermoFisher Scientific) by incubation at 4 °C for 1 h. The beads were then washed with 3 x 1 mL binding buffer with centrifugation at 1,000 x g , 4 °C, 2 min between each wash.

Techniques: Incubation, SDS Page, Silver Staining, Spectroscopy

Journal: Human molecular genetics

Article Title: Aberrant molecular properties shared by familial Parkinson's disease-associated mutant UCH-L1 and carbonyl-modified UCH-L1.

doi: 10.1093/hmg/ddn037

Figure Lengend Snippet: Figure 1. Aberrant biochemical properties of mutant I93M UCH-L1. [(A)–(D)] SH-SY5Y (A) and (C), Neuro2a (B) and COS-7 cells (D) were transfected with the indicated constructs. Forty-eight hours after transfection, soluble and insoluble fractions were prepared and analyzed by immunoblotting. [(E)–(G)] COS-7 cells were transfected with the indicated constructs. Cell lysates were immunoprecipitated using anti-FLAG antibody and analyzed by silver staining [(E) and (F)] or by immunoblotting (G). In the presence of FLAG-tagged UCH-L1, UCH-L1-interacting proteins were co-immunoprecipitated with UCH-L1 [(E), lane 2], whereas in the absence of FLAG-tagged UCH-L1, proteins were non-specifically precipitated with anti-FLAG beads [(E), lane 1]. Mono ub, monoubiquitin (G).

Article Snippet: For the immunoprecipitation of endogenous UCH-L1 (Fig. 5D), 100 mg anti-UCH-L1 antibody (53) or 100 mg normal rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was immobilized to 100 ml of protein G beads using a Seize X Protein G Immnoprecipitation Kit (Pierce, Rockford, IL, USA).

Techniques: Mutagenesis, Transfection, Construct, Western Blot, Immunoprecipitation, Silver Staining

Journal: Human molecular genetics

Article Title: Aberrant molecular properties shared by familial Parkinson's disease-associated mutant UCH-L1 and carbonyl-modified UCH-L1.

doi: 10.1093/hmg/ddn037

Figure Lengend Snippet: Figure 2. Abnormal biochemical properties of carbonyl-modified UCH-L1. (A) COS-7 cells transfected with FLAG-tagged UCH-L1WT were treated with or without the indicated concentrations of carbonyl compounds for 90 min, and immunoprecipitation was performed using anti-FLAG antibody. To detect carbonyl- modified UCH-L1, immunoprecipitants were derivatized with DNPH and immunoblotted using anti-DNP or anti-UCH-L1 antibodies. [(B), (E), (H) and (I)] COS-7 cells transfected with FLAG-tagged UCH-L1WT were treated with the indicated concentrations of HNE [(B) and (E)], HHE (H) or 2-propenal (I) for 90 min, and immunoprecipitation was performed using anti-FLAG antibody. Immunoprecipitants were analyzed by immunoblotting or by silver staining. [(C), (F) and (G)] COS-7 cells transfected with FLAG-tagged UCH-L1WT were treated with the indicated concentrations of HNE (C), HHE (F) or 2-propenal (G). Soluble and insoluble fractions were analyzed by immunoblotting. (D) COS-7 cells transfected with the indicated constructs were treated with or without HNE, and insoluble fractions were prepared. Immunoblotting shows that the insoluble UCH-L1 that is accumulated upon HNE treatment is modified by HNE.

Article Snippet: For the immunoprecipitation of endogenous UCH-L1 (Fig. 5D), 100 mg anti-UCH-L1 antibody (53) or 100 mg normal rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was immobilized to 100 ml of protein G beads using a Seize X Protein G Immnoprecipitation Kit (Pierce, Rockford, IL, USA).

Techniques: Transfection, Immunoprecipitation, Western Blot, Silver Staining, Construct

Journal: Human molecular genetics

Article Title: Aberrant molecular properties shared by familial Parkinson's disease-associated mutant UCH-L1 and carbonyl-modified UCH-L1.

doi: 10.1093/hmg/ddn037

Figure Lengend Snippet: Figure 3. Susceptibility of UCH-L1 to HNE modification and structural prop- erties of UCH-L1 variants. (A) COS-7 cells transfected with the indicated con- structs were treated with or without 100 mM HNE and analyzed by immunoblotting and CBB staining. (B) COS-7 cells transfected with the indi- cated constructs were treated with 100 mM of HNE, and immunoprecipitation was performed using anti-FLAG antibody. Immunoprecipitants were analyzed by immunoblotting. [(C) and (D)] Structural model for human UCH-L1. Cys-90, Cys-152 and Cys-220 sidechains are shown in magenta, and back- bones are shown in blue (C), using Cn3D software (version 4.1) and NCBI’s structural model (mmdbId:38174). Cys-132 and Cys-152 sidechains are shown in magenta, and backbones are shown in blue (D). (E) CD spectra (mean residue ellipticity) for recombinant human UCH-L1 proteins. Wild-type UCH-L1 is shown in red and HNE-modified UCH-L1 in blue. (F) HNE modification of the recombinant UCH-L1 used in (E) was analyzed by immunoblotting. Modification of UCH-L1 by HNE was detected.

Article Snippet: For the immunoprecipitation of endogenous UCH-L1 (Fig. 5D), 100 mg anti-UCH-L1 antibody (53) or 100 mg normal rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was immobilized to 100 ml of protein G beads using a Seize X Protein G Immnoprecipitation Kit (Pierce, Rockford, IL, USA).

Techniques: Transfection, Western Blot, Staining, Construct, Immunoprecipitation, Software, Circular Dichroism, Residue, Recombinant

Journal: Human molecular genetics

Article Title: Aberrant molecular properties shared by familial Parkinson's disease-associated mutant UCH-L1 and carbonyl-modified UCH-L1.

doi: 10.1093/hmg/ddn037

Figure Lengend Snippet: Figure 4. Cysteine residues of UCH-L1 modified by HAE. [(A), (C), (D), (F) and (G)] COS-7 cells transfected with the indicated constructs were treated with or without 100 mM HNE or HHE. Immunoprecipitation was performed using anti-FLAG antibody, and immunoprecipitants were analyzed by immunoblotting or by silver staining. [(B) and (E)] COS-7 cells transfected with the indicated constructs were treated with or without 100 mM HNE or HHE. Soluble and insoluble fractions were analyzed by immunoblotting.

Article Snippet: For the immunoprecipitation of endogenous UCH-L1 (Fig. 5D), 100 mg anti-UCH-L1 antibody (53) or 100 mg normal rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was immobilized to 100 ml of protein G beads using a Seize X Protein G Immnoprecipitation Kit (Pierce, Rockford, IL, USA).

Techniques: Transfection, Construct, Immunoprecipitation, Western Blot, Silver Staining

Journal: Human molecular genetics

Article Title: Aberrant molecular properties shared by familial Parkinson's disease-associated mutant UCH-L1 and carbonyl-modified UCH-L1.

doi: 10.1093/hmg/ddn037

Figure Lengend Snippet: Figure 5. Physical interactions of UCH-L1 with tubulin. (A) Lysates of HeLa cells transfected with the indicated constructs (control: GFP) were immunopre- cipitated with anti-FLAG antibody and analyzed by silver staining. Proteins 50 kDa in size were subjected to LC-MS/MS analysis. (B) Lysates of COS-7 cells transfected with the indicated constructs (control: empty vector) were immunoprecipitated with anti-FLAG antibody and analyzed by immunoblotting. (C) Lysates of NIH-3T3 cells stably expressing FLAG-HA-tagged UCH-L1 were immunoprecipitated with the indicated antibodies and analyzed by immunoblotting. (D) Lysates of Neuro2a cells were immunoprecipitated with control IgG or anti-UCH-L1 antibody and analyzed by immunoblotting. –IgG, without IgG. (E) A pull-down assay was performed using the indicated purified proteins. [(F)–(I)] COS-7 cells transfected with the indicated constructs were treated with the indi- cated concentrations of HNE. Lysates were immunoprecipitated with anti-FLAG antibody and analyzed by immunoblotting.

Article Snippet: For the immunoprecipitation of endogenous UCH-L1 (Fig. 5D), 100 mg anti-UCH-L1 antibody (53) or 100 mg normal rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was immobilized to 100 ml of protein G beads using a Seize X Protein G Immnoprecipitation Kit (Pierce, Rockford, IL, USA).

Techniques: Transfection, Construct, Control, Silver Staining, Liquid Chromatography with Mass Spectroscopy, Plasmid Preparation, Immunoprecipitation, Western Blot, Stable Transfection, Expressing, Pull Down Assay

Journal: Human molecular genetics

Article Title: Aberrant molecular properties shared by familial Parkinson's disease-associated mutant UCH-L1 and carbonyl-modified UCH-L1.

doi: 10.1093/hmg/ddn037

Figure Lengend Snippet: Figure 6. Effects of the I93M mutation and HNE modification of UCH-L1 on tubulin polymerization. [(A) and (B)] A tubulin polymerization assay was per- formed in the absence (control) or in the presence of recombinant UCH-L1. The assays were performed at least three times; representative results are shown. [(C) and (D)] Total lysates (C), soluble tubulin fractions and polymeric tubulin fractions (D) of NIH-3T3 cells stably expressing FLAG-HA-tagged UCH-L1 were analyzed by immunoblotting. (E) Interactions of proteins with microtubules. After the tubulin polymerization assay, the polymerized tubulin was pelleted by centrifugation. The indicated volumes of samples from the supernatants (S) and the pellets (P) were analyzed by CBB staining. BSA was used as a control that does not specifically interact with microtubules. The amount of BSA detected in the pellet fraction was approximately one-twelfth of the amount detected in the supernatant fraction. UCH-L1 levels in the pellet fraction were below detectable levels. (F) Differentiated Neuro2a cells transfected with the indicated constructs were incubated with or without 5 mM paclitaxel for 24 h. Cell death was assessed by a lactate dehydrogenase release assay. Data are expressed as the means+SD (n ¼ 4). P , 0.01 (t-test).

Article Snippet: For the immunoprecipitation of endogenous UCH-L1 (Fig. 5D), 100 mg anti-UCH-L1 antibody (53) or 100 mg normal rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was immobilized to 100 ml of protein G beads using a Seize X Protein G Immnoprecipitation Kit (Pierce, Rockford, IL, USA).

Techniques: Mutagenesis, Polymerization Assay, Control, Recombinant, Stable Transfection, Expressing, Western Blot, Centrifugation, Staining, Transfection, Construct, Incubation, Release Assay

Journal: Human molecular genetics

Article Title: Aberrant molecular properties shared by familial Parkinson's disease-associated mutant UCH-L1 and carbonyl-modified UCH-L1.

doi: 10.1093/hmg/ddn037

Figure Lengend Snippet: Figure 7. Amino acid residues of UCH-L1 involved in the interaction with tubulin. (A) Alanine-scanning mutagenesis of UCH-L1. Lysates of COS-7 cells transfected with the indicated constructs were immunoprecipitated with anti-FLAG antibody and analyzed by immunoblotting. (B) Structural model for human UCH-L1. Cys-90 is shown in blue, Arg-63 and His-185 are in magenta and basic and acidic amino acid residues that had no effect on tubulin interaction (Figs 5B and7A) are shown in white, using NCBI’s structural model (mmdbId:38174). (C) A tubulin polymerization assay was performed in the absence (control) or in the presence of recombinant UCH-L1. (D) Schematic representation of a model for the roles of UCH-L1I93M and carbonyl-modified UCH-L1 in PD. The I93M mutation (as occurs in familial PD associated with UCH-L1I93M) and carbonyl modification (as occurs in sporadic PD) cause conformational changes in UCH-L1. Owing to the excess of oxidative stresses including HNE (in the case of sporadic PD) and the abundant expression of UCH-L1 in dopa- minergic neurons, abnormal UCH-L1 proteins are overproduced in dopaminergic neurons. Abnormal UCH-L1 interacts with tubulin and aberrantly modulates tubulin polymerization. The aberrant interactions of UCH-L1 variants with multiple proteins may also cause dysfunctions of interacting proteins. The deregula- tions of abnormal UCH-L1-interacting proteins, including tubulin, result in dysfunction of dopaminergic neurons, leading to neurodegeneration.

Article Snippet: For the immunoprecipitation of endogenous UCH-L1 (Fig. 5D), 100 mg anti-UCH-L1 antibody (53) or 100 mg normal rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was immobilized to 100 ml of protein G beads using a Seize X Protein G Immnoprecipitation Kit (Pierce, Rockford, IL, USA).

Techniques: Mutagenesis, Transfection, Construct, Immunoprecipitation, Western Blot, Polymerization Assay, Control, Recombinant, Expressing