Journal: Nature Communications

Article Title: A neurodegeneration checkpoint mediated by REST protects against the onset of Alzheimer’s disease

doi: 10.1038/s41467-023-42704-6

Figure Lengend Snippet: a Cultured human SHY-5Y neuroblastoma cells were transduced with recombinant lentiviruses leading to either REST inhibition (short hairpin RNA; sh4) or overexpression (human REST cDNA: hREST ), as previously described . Expression of γ-secretase components was assessed by qRT-PCR. b REST lx/lx MEF cells were transduced with Cre recombinase (generating REST-KO lines 1–3) or a control vector without Cre (lines WT 1–3). The REST floxed ( REST lx ) and Cre-recombined REST ( REST rec ) alleles were detected by PCR. No REST lx band was detected in REST-KO cells, suggesting complete Cre -mediated recombination. c qRT-PCR using two sets of primers spanning the transcript shows loss of REST mRNA in REST-KO MEFs. d Immunolabeling with anti-REST antibody (REST14 ; white) shows loss of REST expression in REST-KO MEFs. Nuclei are labeled with DAPI (blue). Scale bar, 40 μm. e Western blot analysis of γ-secretase components in WT and REST-KO MEFs. The transferrin receptor (TfR) served as loading control. f Quantification of protein levels normalized to TfR shown as percentage expression in REST-KO relative to WT cells (interrupted line: 100%). For PS1, similar results were seen with antibodies against the N-terminus (NTF; antibody 231f) or C-terminus (CTF; antibody EP2000Y). g REST-KO MEFs show elevated γ-secretase enzymatic activity. Solubilized membranes were incubated with Met-C99-FLAG in the presence or absence of a γ-secretase inhibitor (+I), and levels of AICD-FLAG were determined by Western blotting using TfR as a loading control. h Quantification of γ-secretase activity in membrane preparations from WT and REST-KO cells. AICD-FLAG levels were normalized to TfR, and shown as percentage expression in REST-KO relative to WT cells (interrupted line: 100%). Loss of REST in REST-KO MEFs leads to elevated Aβ40 ( i ) and Aβ42 ( j ) levels following transfection of hAPP WT or hAPP Swe . Lentiviral transduction of human REST cDNA (hREST) suppresses Aβ production. Individual values and the mean ± S.E.M are shown for n = 6 ( a ) or n = 3 ( c , f , h , i , j ) independent experiments. P -values are derived from two-tailed unpaired t -tests ( a , c , f , h ) or one-way ANOVA with Tukey’s post-hoc test ( i , j ). Source data are provided as a Source Data file.

Article Snippet: Additional primary antibodies were as follows: anti-human Aβ rabbit monoclonal IgG antibody (Cell Signaling, Cat. No. 8243); anti-human APP mouse monoclonal IgG antibody (clone 6E10; Covance, Catalog No. SIG-39320); anti-actin mouse monoclonal IgG antibody (clone ACTN05 (C4); ThermoFisher Scientific, Catalog No. MA5-11869); anti-NeuN mouse monoclonal IgG antibody (clone A60, Millipore, MAB377); anti-MAP2 goat polyclonal IgG antibody (PhosphoSolutions, Catalog. No. 1099-MAP2); anti-CDK5 mouse monoclonal IgG antibody (clone 4E4; Novus Bio, Catalog No. NBP2-37602); anti-GSK3β mouse monoclonal IgG antibody (clone D5C5Z; Novus Bio, catalog No. NBP1-47470S); anti-PS1 C-terminal (CTF) rabbit monoclonal IgG antibody (clone EP2000Y; Abcam, Catalog No. ab76083); anti-PS1 N-terminal (NTF) rabbit polyclonal IgG antibody (231-f; made in the Yankner lab); anti-Nicastrin mouse monoclonal IgG antibody (clone 9C3; Biolegend, Catalog No. 852301); anti-Nicastrin rabbit polyclonal IgG antibody (Sigma Millipore, Catalog No. N1660); anti-PEN2 rabbit polyclonal IgG antibody (ProScience, Catalog No.3981); anti-PEN2 rabbit monoclonal IgG antibody (clone EPR9200; Abcam, Catalog No. ab154830); anti-PEN2 rabbit polyclonal IgG (ProScience, Catalog No. 3981); anti-Transferrin receptor mouse monoclonal IgG antibody (clone H68.4; ThermoFisher Scientific, Catalog No. 13-6800); anti-BiP/GRP78 mouse monoclonal IgG (clone C38; ThermoFisher Scientific, clone C38, Catalog No. 14-9768-82); anti-β-catenin goat polyclonal IgG (R&D Systems, Catalog No. AF1329); non-specific rabbit IgG antibody (Sigma Millipore, Catalog No. 17-641); and anti-FLAG mouse monoclonal IgG1 antibody (clone M2; Sigma Millipore, Catalog No. F3165).

Techniques: Cell Culture, Transduction, Recombinant, Inhibition, shRNA, Over Expression, Expressing, Quantitative RT-PCR, Control, Plasmid Preparation, Immunolabeling, Labeling, Western Blot, Activity Assay, Incubation, Membrane, Transfection, Derivative Assay, Two Tailed Test

Journal: eLife

Article Title: Predicted glycosyltransferases promote development and prevent spurious cell clumping in the choanoflagellate S. rosetta

doi: 10.7554/eLife.41482

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pEFl5'-Actin3'::pac-P2A-couscous-mTFP, , , this paper , Addgene ID NK698 , Parent vector: pEFl5'- Actin3'::pac-P2A-mTFP ; couscous inserted using Gibson assembly.

Techniques: Recombinant, Marker, Plasmid Preparation

Journal: Molecular Cell

Article Title: Histone H2AK119 Mono-Ubiquitination Is Essential for Polycomb-Mediated Transcriptional Repression

doi: 10.1016/j.molcel.2019.11.021

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-Mtf2 , Proteintech , Cat# 16208-1-AP; RRID: AB_2147370.

Techniques: Control, Virus, Recombinant, Transfection, Purification, Extraction, Western Blot, Plasmid Preparation, Software

Journal: eLife

Article Title: Predicted glycosyltransferases promote development and prevent spurious cell clumping in the choanoflagellate S. rosetta

doi: 10.7554/eLife.41482

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pEFl5'-Actin3'::pac-P2A-mTFP-couscous , , this paper , Addgene ID NK700 , Parent vector: pEFl5'-Actin3':: pac-P2A-mTFP ; couscous inserted using Gibson assembly.

Techniques: Recombinant, Marker, Plasmid Preparation

Journal: The Journal of Experimental Medicine

Article Title: Iron modulation of erythropoiesis is associated with Scribble-mediated control of the erythropoietin receptor

doi: 10.1084/jem.20170396

Figure Lengend Snippet: TfR2 stability is regulated by iron, isocitrate, and cathepsin activity. (A) Immunoblot of whole cell lysates from human progenitors cultured in iron-replete (100% TSAT) or -deficient (15% TSAT) erythroid medium ± isocitrate and treated with cycloheximide (CHX). (B) Densitometry for fraction of residual TfR2 at 3 versus 0 h of cycloheximide, with normalization to TfR1 ( n = 3, one-way ANOVA). IC, isocitrate. (C) Immunoblot of membrane fractions from progenitors cultured in erythroid medium with indicated TSATs ± the cathepsin inhibitor CA074me. Representative results from three independent experiments (Fig. S4 A). (D) Plots from flow cytometry of progenitors cultured in erythroid medium with indicated TSATs ± cathepsin inhibitor. Graph depicts percent decrease in GPA expression associated with iron deprivation ± cathepsin inhibition ( n = 3, Student’s t test). (E) IP of endogenous TfR1 and TfR2 using extracts from K562 cells untreated (Unt) or cultured with DFO or with FA followed by immunoblot detection. Right, input immunoblot. Representative results from three independent experiments. (F) IP of endogenous EpoR and TfR2 from extracts of HUDEP-2 cells cultured ± overnight iron withdrawal followed by immunoblot detection. Right, input immunoblot. Representative results from two independent experiments. IB, immunoblot. Graphs depict mean ± SEM from the indicated number of independent experiments. *, P < 0.05; ***, P < 0.001.

Article Snippet: Immunoblot primary antibodies consisted of the following: rabbit polyclonal anti-Scribble (4475; Cell Signaling Technology), mouse monoclonal anti–human TfR2 (sc-32271; Santa Cruz Biotechnology), rabbit polyclonal anti-EpoR (sc-695; Santa Cruz Biotechnology), rabbit polyclonal anti-TfR1 (sc-9099; Santa Cruz Biotechnology), rabbit polyclonal anti-ATP1A1 (3010; Cell Signaling Technology), rabbit polyclonal anti-STAT5 (9363S; Cell Signaling Technology), rabbit monoclonal antiphospho-STAT5 (pY694; 4322P; Cell Signaling Technology), rabbit monoclonal antiphospho-Akt (pS473; 4060S; Cell Signaling Technology), rabbit monoclonal anti-Akt (4685S; Cell Signaling Technology), mouse monoclonal antitubulin (clone DM1A; T9026; Sigma-Aldrich), and rabbit polyclonal anti-LDH (sc-33781; Santa Cruz Biotechnology).

Techniques: Activity Assay, Western Blot, Cell Culture, Membrane, Flow Cytometry, Expressing, Inhibition

Journal: The Journal of Experimental Medicine

Article Title: Iron modulation of erythropoiesis is associated with Scribble-mediated control of the erythropoietin receptor

doi: 10.1084/jem.20170396

Figure Lengend Snippet: TfR2 regulates Scribble and the iron deprivation response. (A) Immunoblot of whole cell lysates from progenitors transduced with control or TfR2-targeting lentiviral constructs and cultured in iron-replete erythroid medium. (B) Graph of densitometry from multiple experiments as in A, reflecting 3.4-fold increase in levels of Scribble associated with TfR2 knockdown, with normalization to tubulin (Tub; n = 3, one-way ANOVA). (C) Immunoblot of membrane fractions from progenitors transduced with control or TfR2-targeting lentiviral shRNA constructs and cultured in erythroid medium with indicated TSATs ± cathepsin inhibitor. (D) Graphs of densitometry from multiple experiments as in C, comparing the impact of cathepsin inhibition on Scribble levels in progenitors ± TfR2 knockdown and iron deprivation as indicated, with normalization to total Ponceau signal ( n = 3, Student’s t test). CA, cathepsin. (E) Immunoblot of membrane fractions from progenitors transduced with control or TfR2-targeting lentiviral shRNA constructs and cultured in erythroid medium with indicated TSATs ± isocitrate. (F) Graph of densitometry from multiple experiments as in E for relative Scribble levels, with normalization to total Ponceau signal ( n = 3, two-way ANOVA). (G) Flow cytometry of progenitors transduced with control or TfR2-targeting lentiviral shRNA constructs and cultured in erythroid medium with indicated TSATs ± isocitrate. (H) Graphic summary of multiple flow cytometry studies as in G, showing fold change in the percentage of GPA+ cells normalized to empty vector–transduced cells cultured in medium with 100% TSAT, fold change in the GPA percentage associated with iron deprivation, and fold increase in the GPA percentage associated with isocitrate treatment of iron-deprived cells ( n = 4; left, two-way ANOVA; right, Student’s t test). EV, control; IC, isocitrate; shTfR2, TfR2-targeting lentiviral shRNA constructs. Graphs depict mean ± SEM from the indicated number of independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant.

Article Snippet: Immunoblot primary antibodies consisted of the following: rabbit polyclonal anti-Scribble (4475; Cell Signaling Technology), mouse monoclonal anti–human TfR2 (sc-32271; Santa Cruz Biotechnology), rabbit polyclonal anti-EpoR (sc-695; Santa Cruz Biotechnology), rabbit polyclonal anti-TfR1 (sc-9099; Santa Cruz Biotechnology), rabbit polyclonal anti-ATP1A1 (3010; Cell Signaling Technology), rabbit polyclonal anti-STAT5 (9363S; Cell Signaling Technology), rabbit monoclonal antiphospho-STAT5 (pY694; 4322P; Cell Signaling Technology), rabbit monoclonal antiphospho-Akt (pS473; 4060S; Cell Signaling Technology), rabbit monoclonal anti-Akt (4685S; Cell Signaling Technology), mouse monoclonal antitubulin (clone DM1A; T9026; Sigma-Aldrich), and rabbit polyclonal anti-LDH (sc-33781; Santa Cruz Biotechnology).

Techniques: Western Blot, Transduction, Control, Construct, Cell Culture, Knockdown, Membrane, shRNA, Inhibition, Flow Cytometry, Plasmid Preparation

Journal: The Journal of Experimental Medicine

Article Title: Iron modulation of erythropoiesis is associated with Scribble-mediated control of the erythropoietin receptor

doi: 10.1084/jem.20170396

Figure Lengend Snippet: Blockade of isocitrate production impairs TfR2 surface delivery. (A) Pulse-chase analysis of surface-biotinylated TfR2 in erythroid progenitors, with assessment of the effects of aconitase inhibition. Cells cultured in erythroid medium ± 50 µM FA underwent surface biotinylation and were returned to culture for the indicated durations. The cells were then harvested for streptavidin pull-down and immunoblot. (B) Graph of densitometry from multiple experiments as in A for relative TfR2 levels, with normalization to TfR1 ( n = 3, no intergroup difference by two-way ANOVA). (C) Analysis of the surface delivery of TfR2, with assessment of the effects of aconitase inhibition. Cells cultured in erythroid medium ± FA underwent trypsin-mediated stripping of surface TfR2 followed by recovery culture for 0–60 min. The cells were then harvested for streptavidin pull-down and immunoblot. Also shown are control cells not subjected to trypsinization (−Tryp). (D) Graph of densitometry from multiple experiments similar to C for surface TfR2 levels at 0–3 h after trypsinization normalized to total biotinylated protein ( n = 4). (E) Quantitation from multiple experiments as in C of the effect of FA treatment on surface TfR2 recovery at 10 min after trypsinization, showing TfR2 levels normalized to total biotinylated protein and fold change in normalized TfR2 from 0 to 10 min of recovery ( n = 3, Student’s t test, *, P < 0.05). Graphs depict mean ± SEM from the indicated number of independent experiments.

Article Snippet: Immunoblot primary antibodies consisted of the following: rabbit polyclonal anti-Scribble (4475; Cell Signaling Technology), mouse monoclonal anti–human TfR2 (sc-32271; Santa Cruz Biotechnology), rabbit polyclonal anti-EpoR (sc-695; Santa Cruz Biotechnology), rabbit polyclonal anti-TfR1 (sc-9099; Santa Cruz Biotechnology), rabbit polyclonal anti-ATP1A1 (3010; Cell Signaling Technology), rabbit polyclonal anti-STAT5 (9363S; Cell Signaling Technology), rabbit monoclonal antiphospho-STAT5 (pY694; 4322P; Cell Signaling Technology), rabbit monoclonal antiphospho-Akt (pS473; 4060S; Cell Signaling Technology), rabbit monoclonal anti-Akt (4685S; Cell Signaling Technology), mouse monoclonal antitubulin (clone DM1A; T9026; Sigma-Aldrich), and rabbit polyclonal anti-LDH (sc-33781; Santa Cruz Biotechnology).

Techniques: Pulse Chase, Inhibition, Cell Culture, Western Blot, Stripping Membranes, Control, Quantitation Assay

Journal: The Journal of Experimental Medicine

Article Title: Iron modulation of erythropoiesis is associated with Scribble-mediated control of the erythropoietin receptor

doi: 10.1084/jem.20170396

Figure Lengend Snippet: A schematic model for erythroid coupling of iron availability and Epo responsiveness. Under conditions of high iron (top left), TfR2-associated vesicles traffic predominantly to the cell surface and minimally to lysosomes. Lysosomal catabolism of TfR2–Scribble complexes occurs at a low, basal rate, permitting maintenance of critical Scribble levels. At these levels, Scribble promotes efficient EpoR surface presentation. Under conditions of low iron (top right) or aconitase inhibition, TfR2-associated vesicles traffic predominantly to lysosomes, accelerating catabolism of TfR2–Scribble complexes. As a result, Scribble levels fall below a critical threshold, impairing surface delivery of EpoR. The bottom two panels depict signaling configurations in each condition. With high iron (bottom left), high surface EpoR levels enable robust STAT5 activation, but abundant surface Scribble blunts Akt activation. With low iron (bottom right), Scribble deficiency decreases surface EpoR levels, leading to diminished STAT5 activation despite rising serum Epo levels. Akt signaling, in contrast, is preserved or even enhanced because of its liberation from Scribble inhibition. MVB, multivesicular body.

Article Snippet: Immunoblot primary antibodies consisted of the following: rabbit polyclonal anti-Scribble (4475; Cell Signaling Technology), mouse monoclonal anti–human TfR2 (sc-32271; Santa Cruz Biotechnology), rabbit polyclonal anti-EpoR (sc-695; Santa Cruz Biotechnology), rabbit polyclonal anti-TfR1 (sc-9099; Santa Cruz Biotechnology), rabbit polyclonal anti-ATP1A1 (3010; Cell Signaling Technology), rabbit polyclonal anti-STAT5 (9363S; Cell Signaling Technology), rabbit monoclonal antiphospho-STAT5 (pY694; 4322P; Cell Signaling Technology), rabbit monoclonal antiphospho-Akt (pS473; 4060S; Cell Signaling Technology), rabbit monoclonal anti-Akt (4685S; Cell Signaling Technology), mouse monoclonal antitubulin (clone DM1A; T9026; Sigma-Aldrich), and rabbit polyclonal anti-LDH (sc-33781; Santa Cruz Biotechnology).

Techniques: Inhibition, Activation Assay

Journal: European Journal of Human Genetics

Article Title: An MTF1 binding site disrupted by a homozygous variant in the promoter of ATP7B likely causes Wilson Disease

doi: 10.1038/s41431-018-0221-4

Figure Lengend Snippet: Genomic context of the candidate causative variant. a UCSC Genome Browser (hg19 minus strand) view of the region near the candidate causative variant positioned 676 bp upstream of the canonical ATP7B translation start site (chr13:g.52,586,149A>G, cyan highlight and asterisk). The variant lies in a 100-way vertebrate alignment conserved element and region of high cross-species sequence conservation as computed by PhastCons and PhyloP [30]. The chromatin surrounding chr13:g.52,586,149T>C is hypersensitive to DNaseI (and, therefore, open and accessible) in HepG2 cells. The sequence cloned into the luciferase reporter vector is indicated by the black bar. b Genotype verification for chr13:g.52,586,149T>C (red asterisk) by Sanger sequencing. Representative chromatograms show the indicated number (n=x/y) of amplification products containing the reference (T) or alternate (C) allele for each individual. Box indicates a predicted binding site for MTF1. c The candidate causative variant (bolded, pink) in the Wilson Disease (WD) patient disrupts a key base in the MTF1 position weight matrix. This exact base (boxed) is unaltered in all primates and in dozens of other mammals, even as distant as Tasmanian devil (Supplementary Fig. 1b) (color figure online).

Article Snippet: ChIP-qPCR Polyclonal rabbit anti-MTF1 antibody (Proteintech, Rosemont, IL, USA: 25383-1-AP) was used for chromatin immunoprecipitation.

Techniques: Variant Assay, Sequencing, Clone Assay, Luciferase, Plasmid Preparation, Amplification, Binding Assay

Journal: European Journal of Human Genetics

Article Title: An MTF1 binding site disrupted by a homozygous variant in the promoter of ATP7B likely causes Wilson Disease

doi: 10.1038/s41431-018-0221-4

Figure Lengend Snippet: Functional interrogation of chr13:g.52,586,149T>C. a Luciferase reporter assays in HepG2 cells were performed to quantify differences in transactivation by a 379 bp fragment of the ATP7B promoter with the chr13:g.52,586,149T (Ref) or chr13:g.52,586,149C (Alt) allele, in the presence (+) or absence (−) of MTF1 overexpression (OE). Both with and without MTF1 OE, the reference allele drove significantly higher expression compared to the alternate allele (comparison 1 and 2, respectively). Both alleles experienced dramatic increases in activity with MTF1 OE (comparisons 3 and 4), but the reference allele yielded a greater increase in expression than did the alternate allele in this context (comparison 5). Bars represent mean ± SD. Two-tailed p-values from unpaired t-tests for each comparison are shown above the associated bracket. b ChIP-qPCR was performed to determine the extent of MTF1 binding at the SNV locus (ATP7B) compared to a computationally predicted negative control (Neg Ctrl, in an intron of WDPCP) and to a previously published [32] experimentally validated locus (Pos Ctrl, in the 5′ UTR of SELENOH). Enrichment of DNA immunoprecipitated by MTF1-specific vs. non-specific isotype-matched control antibodies was measured in technical triplicate by quantitative PCR analysis. Bars represent mean ± SD. One-tailed p-values from unpaired t-tests for each comparison are shown above the associated bracket. c Proposed model for disease-causing mechanism of chr13:g.52,586,149T>C: (1) Excess intracellular accumulation of copper, Cu2+, increases (2) expression or nuclear translocation of MTF1 [12, 13]. In wildtype individuals, MTF1 then binds at chr13:g.52,586,149T (3) to recruit transcriptional machinery for upregulating ATP7B expression [34] and eliminating copper through serum ceruloplasmin and, ultimately, through the bile. We propose that the Wilson Disease (WD) patient’s homozygous single nucleotide promoter variant chr13:g.52,586,149T>C exhibits reduced affinity to MTF1. (4) The result is an insufficient ATP7B transcriptional response, consequent copper accumulation, and symptoms characteristic of Wilson Disease

Article Snippet: ChIP-qPCR Polyclonal rabbit anti-MTF1 antibody (Proteintech, Rosemont, IL, USA: 25383-1-AP) was used for chromatin immunoprecipitation.

Techniques: Functional Assay, Luciferase, Over Expression, Expressing, Comparison, Activity Assay, Two Tailed Test, ChIP-qPCR, Binding Assay, Negative Control, Immunoprecipitation, Control, Real-time Polymerase Chain Reaction, One-tailed Test, Translocation Assay, Variant Assay