Journal: bioRxiv

Article Title: A conserved function of corepressors is to nucleate assembly of the transcriptional preinitiation complex

doi: 10.1101/2024.04.01.587599

Figure Lengend Snippet: TPL interacts with the SPT4/SPT5/SPT6 complex through the conserved elongation factor SPT4 A. Venn diagram intersection of genes identified by APEX labelling and R-SGA analysis. 531 nuclear proteins from APEX (teal), 238 up-regulated R-SGA mutants (gold), and 85 down-regulated (red) mutants. B. A nested circle diagram depicts the APEX enrichment of the 40 proteins from the overlapping results of APEX and Up regulated R-SGA. Nested circles represent the relative enrichment positions of known controls (TF-ARF, F-Box-AFB2, TPLN188). Five proteins were enriched at or above the level of the TPLN188-TPLN188 interaction (SPT5, TEF4, RPB2, CDC48, and DED1). C. STRING protein network of the top 40 interacting proteins. The network is enriched for three main categories as defined by GO terminology: proteasome/protein stability - orange, regulation of transcription - yellow, and RNA processing - red. The five most highly enriched by APEX-labelling are highlighted with a heavy weighed outline. The network was clustered to a MCL inflation parameter of 3. Subclusters of genes are colored, and network edges between clusters are shown as dotted lines. Line thickness indicates the strength of support. STRING minimum required interaction score – 0.4. Small circles at the top left of the node indicate whether this R-SGA mutant was validated by flow cytometry, and in which TPL-truncation it was validated. D-G. Cytoplasmic split-ubiquitin system (CytoSUS) assays with candidate interacting proteins. Nub-3xHA is the N-terminal fragment of ubiquitin expressed with no fusion protein and is used as a negative control. -WL, -ADE: dropout lacking Trp, Leu, and Ade (growth control); -WLMH, -ADE: dropout lacking Trp, Leu, His, Met, and Ade (selective media). The plating for each panel was performed at the same day. D. CytoSUS interaction of TPLN188 with yeast proteins identified in the top 40, all individuals enriched for GO terms relating to transcription were tested in addition to other selected candidates. E. CytoSUS interaction of TPLN188 with SPT/DSIF specific components from yeast and Arabidopsis . F . CytoSUS interaction of TPL H1-H2 (LisH domain) with ScSPT4. G. CytoSUS interaction of HsTBL1 N-terminal domain (N98) with ScSPT4. H. Structure of RNA POL II subunits, DNA, PAF complex, with SPT/DSIF proteins – from yeast ( komagataella phaffii ) 7XN7 . Proteins identified by both APEX2 labelling and R- SGA - purples (SPT5, SPT6, RPB2, SPN1), identified only by APEX2 – reds, undetected – gray, SPT4 – pink. I . The TPL-ProteinA-TF fusion protein can pull down TPL, ScSPT4 from yeast extracts using IgG-beads. Detection of the VP16 transcriptional activator demonstrates enrichment of the fusion protein (αVP16). Each prey protein is detected via the 3xHA tag (αHA). J . Bimolecular fluorescence complementation assay performed in Nicotiana benthamiana (tobacco). Each image is an epi-fluorescent micrograph taken at identical magnification from epidermal cells two days post injection. The YFP image is colored green (left panel). p35S:H2B-RFP was used as a control and is false-colored magenta (right panel).

Article Snippet: Cells were then spun down and resuspended in 100LμL of buffer containing fluorescently conjugated antibodies for one hour (5LμL of each antibody per sample): APC-Cy7 Mouse Anti- Human CD4 (1:20 dilution; BD Biosciences, #557871), APC-Cy7 Mouse IgG1, κ Isotype Control (1:20 dilution; BD Biosciences, #557873).

Techniques: Mutagenesis, Flow Cytometry, Negative Control, Bimolecular Fluorescence Complementation Assay, Injection

Journal: bioRxiv

Article Title: An Alzheimer’s disease-associated common regulatory variant in PTK2B has causal effects on microglial function

doi: 10.1101/2023.11.04.565613

Figure Lengend Snippet: Isogenic edited cell lines show that rs28834970 effects PTK2B expression through effects on chromatin accessibility and CEBPβ binding at the intronic enhancer in iPSC-derived macrophages and microglia. A) Boxplots showing PTK2B expression in macrophages harbouring the T/T or C/C allele at rs28834970 measured by RNA-seq (top) and droplet digital q-PCR (ddqPCR) (bottom). Top plot: TPM= transcripts per million. Bottom plot: Relative concentration = copies/ul relative to housekeeping gene (SDHA), n=3 and **p<0.01, one-way paired ANOVA. B) Boxplots showing PTK2B expression in microglia harbouring the T/T or C/C allele at rs28834970 measured by RNA-seq (top) and droplet digital q-PCR (ddqPCR) (bottom). Top plot: TPM= transcripts per million. Bottom plot: Relative concentration = copies/ul relative to housekeeping gene (TBP), n=3 and **p<0.01, one-way paired ANOVA. ns= non- significant C) Coverage tracks of cells with T/T (orange) and C/C (blue) allele at rs28834970. The tracks are: ATAC-seq in macrophages (upper track), ATAC-seq in microglia (second track), CUT&RUN for CEBPβ in microglia (third track) and IgG negative control for CUT&RUN (bottom track). The rs28834970 variant in PTK2B intron is highlighted in yellow. D) Effect of rs28834970 SNP on transcription factor binding probability. Position probability matrices are shown for CEBPβ (top) and CEBPα (middle) as well as the position of the binding motifs on the PTK2B sequence (bottom). The plot was generated using motifbreakerR . E) Volcano plot showing fold changes of CEBPβ binding between microglia harbouring the C/C and the T/T allele for all detectable peaks, measured by CUT&RUN. Blue dots indicate peaks with no significant change while red dots represent significantly increased peaks (logFC>1 and p<0.05). The only significantly increased CEBPβ peak found spans the rs28834970 variant in the PTK2B intron.

Article Snippet: Isotype controls were stained with either 0.2 mg/ml APC-mouse IgG1 k(BD Biosciences), 0.2 mg/ml of PE-mouse IgG2A (BD Biosciences) or 0.2 mg/ml APC/Cy7 mouse IgG1 k (BD Biosciences).

Techniques: Expressing, Binding Assay, Derivative Assay, RNA Sequencing Assay, Concentration Assay, Negative Control, Variant Assay, Sequencing, Generated

Journal: Cell reports

Article Title: Alpha synuclein, the culprit in Parkinson disease, is required for normal immune function

doi: 10.1016/j.celrep.2021.110090

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-mouse APC/Cy7 Hamster IgG1 (clone G235-2356) , BD PharMingen , Cat# 561206.

Techniques: Recombinant, Stripping Membranes, SYBR Green Assay, Multiplex Assay, Software, Flow Cytometry, Light Microscopy

Journal: Developmental and comparative immunology

Article Title: CD9 expression in porcine blood CD4 + T cells delineates two subsets with phenotypic characteristics of central and effector memory cells.

doi: 10.1016/j.dci.2022.104431

Figure Lengend Snippet: Fig. 1. MAb 4H5CR4 recognizes porcine CD9. (A) Reactivity of mAb 4H5CR4 with PBMC subsets: PBMCs were stained with mAbs to CD3, CD4, CD8α, CD8β, γδTCR, CD16, CD21, CD172a, IgM or Siglec-10, and APC-conjugated-goat anti-mouse Ig (y-axis), followed by biotin-conjugated mAb 4H5CR4 and PE-streptavidin (x-axis). Quadrants were set by background staining with irrelevant isotype-matched mAbs. Numbers indicate the percentage of cells within the respective quadrants. Results are representative of at least three independent experiments with cells from 8 to 12-month-old pigs. (B) Molecular characterization: Lysates from alveolar macro phages were resolved by 12% SDS-PAGE under reducing conditions, transferred to nitrocellulose membranes, and probed with mAb 4H5CR4 (1) or an irrelevant isotype-matched mAb (2). Numbers in the right indicate position and size of MW markers. Results are representative of two independent experiments. (C) CHO cells transiently transfected with pCD9-GFP construct were stained with mAb 4H5CR4, or an IgG1 control antibody followed by APC-goat F(ab’)2 anti-mouse Igs and analyzed by flow cytometry. A control staining of non-transfected CHO cells is also shown.

Article Snippet: After a washing step with PBS, cells (106/well) were incubated with mAbs to different surface antigens (2E3, CD4, CD8α, 4H5CR4) for 30 min at 4 ◦C, followed by FITC-conjugated goat anti-mouse IgM, APC-conjugated goat anti-mouse IgG1, PE-Cy7-conjugated goat anti-mouse IgG2a (all from Southern Biotech) and BV421-conjugated goat anti-mouse IgG2b (Jackson B. Álvarez et al. Developmental and Comparative Immunology 133 (2022) 104431 ImmunoResearch Laboratories, Pennsylvania, USA).

Techniques: Staining, SDS Page, Transfection, Construct, Control, Flow Cytometry

Journal: Developmental and comparative immunology

Article Title: CD9 expression in porcine blood CD4 + T cells delineates two subsets with phenotypic characteristics of central and effector memory cells.

doi: 10.1016/j.dci.2022.104431

Figure Lengend Snippet: Fig. 3. CCR7 expression in CD4+ T cell subsets. (A) PBMC were stained with a combination of mAbs to CD4, 2E3, CD8α, and CD9, and recombinant pCCL19-Fc followed by fluorochrome-conjugated secondary goat antibodies specific for mouse Ig isotypes and biotin-conjugated goat anti-human IgG, and then BV421- streptavidin. CD4+ T cells were gated into 2E3+ and 2E3− cells and further subdivided according to CD8α vs CD9 expression. Filled histograms represent the binding of pCCL19-Fc to the gated subsets. Open histograms correspond to the staining with a control human IgG. (B) Median fluorescence intensity of CCR7 expression for each subset. Data of six animals are shown by individual symbols; symbols of same color correspond to the same animal. The mean is indicated by the black bar. Significant differences among groups are denoted by red bars on the top of the graph, p < 0.001.

Article Snippet: After a washing step with PBS, cells (106/well) were incubated with mAbs to different surface antigens (2E3, CD4, CD8α, 4H5CR4) for 30 min at 4 ◦C, followed by FITC-conjugated goat anti-mouse IgM, APC-conjugated goat anti-mouse IgG1, PE-Cy7-conjugated goat anti-mouse IgG2a (all from Southern Biotech) and BV421-conjugated goat anti-mouse IgG2b (Jackson B. Álvarez et al. Developmental and Comparative Immunology 133 (2022) 104431 ImmunoResearch Laboratories, Pennsylvania, USA).

Techniques: Expressing, Staining, Recombinant, Binding Assay, Control, Fluorescence