Journal: Molecular and Cellular Biology

Article Title: A Multifunctional Domain in Human CRM1 (Exportin 1) Mediates RanBP3 Binding and Multimerization of Human T-Cell Leukemia Virus Type 1 Rex Protein

doi: 10.1128/mcb.23.23.8751-8761.2003

Figure Lengend Snippet: FIG. 1. The amino acid residues of hCRM1 required for Rex ac- tivity. (A) The ability of CRM1s to support Rex activity. HeLa cells were transfected with the indicated plasmids. After cell lysis, the amount of CAT and the activity of -Gal were measured and CAT/ -Gal ratios were calculated. The ratio for the control sample without pSRTAgRexM64 and pSRCRM1s was arbitrarily set at 1. The amount of CAT and the -Gal activity in control samples were over 300 pg and 3.0 103 U, respectively. Error bars represent standard deviations. (B) Restoration of Rex activity by overexpression of CRM1s in HeLa cells. The experimental procedure was the same as that described for panel A. (C) Effect of overexpressing CRM1s on Rex activity in REF52 cells. REF52 cells were transfected with the indicated plasmids. At 24 h of posttransfection, CAT/-Gal ratios were calculated. The ratio for the sample without pSRCRM1s was arbi- trarily set at 1. (D) Western blot analysis of various CRM1s. A fraction of each sample used in the experiments described for panel C was subjected to Western blot analysis using the anti-hCRM1 antibody to examine CRM1 protein synthesis. This antibody was raised with the peptide, which represents the carboxy-terminal region of hCRM1 and has a sequence different from that of rCRM1, so it does not recognize endogenous rCRM1. (E) Effect of CRM1s on Rex-mediated Gag expression from HTLV-1 molecular clone. At 48 h posttransfection, Gag/-Gal ratios were calculated. The ratio for the sample without pSRCRM1s was arbitrarily set at 1. (F) A schematic representation of hCRM1 functional domains. The amino acid sequence of hCRM1 is shown. Experiments characterizing the RanGTP binding domain (bro- ken line) (33), LMB binding residue (arrowhead) (24), Rev-interacting amino acids (asterisks) (2), and the domain binding to NES (under- line) (33) have been previously reported. The residues 411, 414, 474, and 481 in hCRM1 are indicated in bold characters, and the corre- sponding residues of rCRM1 are indicated under the hCRM1 se- quence in single-letter amino acid code.

Article Snippet: Mouse anti-GAL4 monoclonal antibody (Santa Cruz Biotechnology) and affinity-purified chicken antihCRM1 antibody (18) were used as primary antibodies to detect GAL-fused proteins and CRM1s, respectively.

Techniques: Activity Assay, Transfection, Lysis, Control, Over Expression, Western Blot, Sequencing, Expressing, Functional Assay, Binding Assay, Residue

Journal: Molecular and Cellular Biology

Article Title: A Multifunctional Domain in Human CRM1 (Exportin 1) Mediates RanBP3 Binding and Multimerization of Human T-Cell Leukemia Virus Type 1 Rex Protein

doi: 10.1128/mcb.23.23.8751-8761.2003

Figure Lengend Snippet: FIG. 2. In vivo interaction of Rex with CRM1 mutants in which one amino acid is replaced. REF52 cells were transfected with the plasmid expressing GAL-CRM1s in combination with pRexVP, pG5BCAT, and pCDM-gal. The cells were harvested and subjected to CAT and -Gal assays, and CAT/-Gal ratios were calculated. The ratio for the control sample, which detected the interaction between GAL-hCRM1 and Rex-VP, was arbitrarily set at 1. The amount of CAT and -Gal activity in control samples were over 400 pg and 3.0 103 U, respec- tively. GAL4 nonfusion protein, expressing only a GAL4 region, was used as a negative control. A fraction of each sample was subjected to Western blot analysis using the anti-GAL4 monoclonal antibody to examine GAL-CRM1 expression.

Article Snippet: Mouse anti-GAL4 monoclonal antibody (Santa Cruz Biotechnology) and affinity-purified chicken antihCRM1 antibody (18) were used as primary antibodies to detect GAL-fused proteins and CRM1s, respectively.

Techniques: In Vivo, Transfection, Plasmid Preparation, Expressing, Control, Activity Assay, Negative Control, Western Blot

Journal: Molecular and Cellular Biology

Article Title: A Multifunctional Domain in Human CRM1 (Exportin 1) Mediates RanBP3 Binding and Multimerization of Human T-Cell Leukemia Virus Type 1 Rex Protein

doi: 10.1128/mcb.23.23.8751-8761.2003

Figure Lengend Snippet: FIG. 6. Binding characterization of two amino acid-substituted CRM1s. (A) In vivo interaction of CRM1s with Rex. REF52 cells were treated as described for Fig. 2 except for the plasmids that expressed two amino acid-substituted CRM1s as a GAL4 fusion protein. A portion of each sample was subjected to Western blot analysis to confirm GAL-CRM1 expression. (B) In vitro binding of h411/414 to RanBP3. As shown in Fig. 5A, in vitro-translated CRM1s were incubated with GST or GST-RanBP3 immobilized on glutathione-Sepharose 4B.

Article Snippet: Mouse anti-GAL4 monoclonal antibody (Santa Cruz Biotechnology) and affinity-purified chicken antihCRM1 antibody (18) were used as primary antibodies to detect GAL-fused proteins and CRM1s, respectively.

Techniques: Binding Assay, In Vivo, Western Blot, Expressing, In Vitro, Incubation

Journal: Cell Reports

Article Title: Fiber deprivation and microbiome-borne curli shift gut bacterial populations and accelerate disease in a mouse model of Parkinson’s disease

doi: 10.1016/j.celrep.2023.113071

Figure Lengend Snippet: Curli-driven phosphorylated alpha-synuclein aggregate formation in fiber-deprived Thy1-Syn14 mice (A) Representative microphotographs of pS129-αSyn-positive structure containing ganglions in the myenteric plexus of WT and TG (left to right) mice of any treatment group (FD PBS to FD EC from top to bottom). Qualitatively, in TG mice, pS129-αSyn-positive structures after FD EC challenges seemed larger and more numerous. Scale bar: 50 μm. (B) Boxplot illustrating area occupied changes by pS129-αSyn-positive structures in ganglions of the myenteric plexus. The most significant area increase of pS129-αSyn-positive structures was seen in TG FD EC-challenged mice. Results were analyzed by Mann-Whitney U test, not corrected for FDR. Sample sizes: WT FD PBS, n = 4; WT FR ΔEC, n = 6; WT FR EC, n = 4; WT FD ΔEC, n = 4; WT FD EC, n = 4; TG FD PBS, n = 4; TG FR ΔEC, n = 5; TG FR EC, n = 7; TG FD ΔEC, n = 5; and TG FD EC, n = 6. See Table S2 .

Article Snippet: The following antibodies were used: monoclonal rabbit anti-pS129-αSyn (Abcam, ab51253; 1:1000), monoclonal mouse anti-pS129-αSyn (Prothena Biosciences Inc., 11A5; 1:1000), polyclonal chicken anti-tyrosine hydroxylase (Abcam, ab76442; 1:1000), polyclonal rabbit anti-tyrosine hydroxylase (Merck (Sigma-Aldrich), AB152; 1:1000), polyclonal rat anti-dopamine transporter (MAB369, Merck (Sigma-Aldrich); 1:1000), polyclonal rabbit pan αSyn (S3062, Sigma-Aldrich, 1:1000), monoclonal mouse human αSyn 211 clone (S5566, Sigma-Aldrich, 1:1000) and rabbit anti-ionized calcium-binding adapter molecule 1 (Iba1) (1919741, Wako, 1:1000).

Techniques: MANN-WHITNEY

Journal: Cell Reports

Article Title: Fiber deprivation and microbiome-borne curli shift gut bacterial populations and accelerate disease in a mouse model of Parkinson’s disease

doi: 10.1016/j.celrep.2023.113071

Figure Lengend Snippet: Curli-driven nigrostriatal alpha-synuclein aggregation and neurodegeneration in Thy1-Syn14 mice was exacerbated by fiber deprivation (A and B) Quantification and representative images of immunofluorescent pS129-αSyn stainings of the (A) dorsal striatum and (B) the SNpc. (A) The pS129-αSyn-positive area occupied was increased by the EC challenge. The FD diet challenge contributed to a lesser extent. The representative images (40×, scale bar: 50 μm) below illustrate the differences in pS129-αSyn accumulations between FR ΔEC- (top) and FR EC- (bottom) challenged TG mice. Sample sizes: FD PBS, n = 4; FR ΔEC, n = 5; FR EC, n = 7; FD ΔEC, n = 8; and FD EC, n = 8. (B) In the SNpc, we distinguished between accumulations in cell bodies (left panel) and other forms (right panel) based on cellular morphology. See for details on quantification. In EC-challenged animals, we found increased pS129-αSyn-positive aggregates. For other structures (see details in the main text), FD exacerbated the EC-induced pathology. The representative images (left: 10× tiles, scale bar: 250 μm; right: zoom in, scale bar: 100 μm) below illustrate the different observed structures of pS129-αSyn-positive structures (see details on the different forms in main text) in FD EC-challenged TG mice. Sample sizes: FD PBS, n = 4; FR ΔEC, n = 5; FR EC, n = 7; FD ΔEC, n = 8; and FD EC, n = 8. (C–E) Quantification and representative images for (C) TH and (D) DAT in the dorsal striatum and (E) TH in the SNpc. (C and D) FD EC transgenic animals exhibited a decrease in axonal and synaptic density. The representative high-magnification (40x, scale bar: 50 μm) images illustrate the differences between FR ΔEC (top row) and FD EC (bottom row) TG mice. Sample sizes: FD PBS, n = 4; FR ΔEC, n = 5; FR EC, n = 8; FD ΔEC, n = 8; and FD EC, n = 8. (E) In the SNpc, curli drove neurodegeneration independently of the diet. FD did however exacerbate the pathology. The representative images (10×; scale bar: 250 μm) illustrate the average differences between FR ΔEC (top) and FD EC (bottom) transgenic animals. Sample sizes: FD PBS, n = 4; FR ΔEC, n = 5; FR EC, n = 7; FD ΔEC, n = 8; and FD EC, n = 8. All results were analyzed by Mann-Whitney U test, not corrected for FDR. See Table S2 .

Article Snippet: The following antibodies were used: monoclonal rabbit anti-pS129-αSyn (Abcam, ab51253; 1:1000), monoclonal mouse anti-pS129-αSyn (Prothena Biosciences Inc., 11A5; 1:1000), polyclonal chicken anti-tyrosine hydroxylase (Abcam, ab76442; 1:1000), polyclonal rabbit anti-tyrosine hydroxylase (Merck (Sigma-Aldrich), AB152; 1:1000), polyclonal rat anti-dopamine transporter (MAB369, Merck (Sigma-Aldrich); 1:1000), polyclonal rabbit pan αSyn (S3062, Sigma-Aldrich, 1:1000), monoclonal mouse human αSyn 211 clone (S5566, Sigma-Aldrich, 1:1000) and rabbit anti-ionized calcium-binding adapter molecule 1 (Iba1) (1919741, Wako, 1:1000).

Techniques: Transgenic Assay, MANN-WHITNEY

Journal: Cell Reports

Article Title: Fiber deprivation and microbiome-borne curli shift gut bacterial populations and accelerate disease in a mouse model of Parkinson’s disease

doi: 10.1016/j.celrep.2023.113071

Figure Lengend Snippet:

Article Snippet: The following antibodies were used: monoclonal rabbit anti-pS129-αSyn (Abcam, ab51253; 1:1000), monoclonal mouse anti-pS129-αSyn (Prothena Biosciences Inc., 11A5; 1:1000), polyclonal chicken anti-tyrosine hydroxylase (Abcam, ab76442; 1:1000), polyclonal rabbit anti-tyrosine hydroxylase (Merck (Sigma-Aldrich), AB152; 1:1000), polyclonal rat anti-dopamine transporter (MAB369, Merck (Sigma-Aldrich); 1:1000), polyclonal rabbit pan αSyn (S3062, Sigma-Aldrich, 1:1000), monoclonal mouse human αSyn 211 clone (S5566, Sigma-Aldrich, 1:1000) and rabbit anti-ionized calcium-binding adapter molecule 1 (Iba1) (1919741, Wako, 1:1000).

Techniques: Plasmid Preparation, Recombinant, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Amplification, Sequencing, Software