Journal: Cell Reports

Article Title: The Arabidopsis F-box protein FBW2 targets AGO1 for degradation to prevent spurious loading of illegitimate small RNA

doi: 10.1016/j.celrep.2022.110671

Figure Lengend Snippet:

Article Snippet: Anti cMyc-HRP , Miltenyi , Cat# 130-092-113, RRID: AB_871937.

Techniques: Virus, Recombinant, SYBR Green Assay, Western Blot, Protease Inhibitor, Isolation, Reverse Transcription, Magnetic Beads, Mass Spectrometry, Cloning, Sequencing, Software

Journal:

Article Title: DNA-PKcs function regulated specifically by protein phosphatase 5

doi: 10.1073/pnas.0307765100

Figure Lengend Snippet: Phosphorylation status of DNA–PKcs after irradiation in PP5 overexpressing clones. PP5.C4 and PP5.C13 subclones were irradiated with IR (11.5 Gy) and recovered for 30–410 min. The undamaged control (UD) is shown on the left. Nuclear extracts were prepared, and 20–60 μg of protein was applied to a SDS gel. After Western blotting, the membrane was stained with rabbit antibody to DNA–PKcs pT2609 (A), rabbit antibody to DNA–PKcs pS2056 (B), or rabbit antibody to ATM pS1981 (C). For loading controls the same membranes were stripped and incubated with antibody to total DNA–PKcs or to DNA polymerase δ. PP5 overexpresssion was visualized by anti-PP5 antibody. * and ** denote phosphorylated DNA–PKcs degradation products.

Article Snippet: The proteins were transferred by Western blotting onto a nitrocellulose membrane (Bio-Rad) and incubated with antibody 18-2 to DNA–PKcs (Neomarkers, Fremont, CA), antibody to pT2609, antibody to pS2056 site, antibody to DNA polymerase δ (Transduction Laboratories, Lexington, KY), antibody to PP5 (Transduction Laboratories), or antibody to ATM pS1981 (Rockland, Gilbertsville, PA).

Techniques: Phospho-proteomics, Irradiation, Clone Assay, Control, SDS-Gel, Western Blot, Membrane, Staining, Incubation

Journal: medRxiv

Article Title: In severe COVID-19, SARS-CoV-2 induces a chronic, TGF-β-dominated adaptive immune response

doi: 10.1101/2020.09.04.20188169

Figure Lengend Snippet: Peripheral blood activated B cells (CD27 high CD38 high ) were isolated and sorted by FACS for single cell sequencing. a) Percentage of CD27 high CD38 high cells among live B cells. Each sample is represented by a circle. The line indicates the median. Unpaired two sided Mann-Whitney U test. b) UMAP representation of 72277 CD27 high CD38 high sorted B cells from 9 COVID-19 ICU patients and 3 healthy controls. Between 2416 and 11229 cells were recovered per sample. Transcriptionally similar clusters were identified using shared nearest neighbor (SNN) modularity optimization (left). The combination of CD19, MS4A1, CD27, CD38, IFIT1, MIKI67, IRF4 and PRDM1 expression were used for annotation of the different activation/differentiation stages (right). c) Heatmap of genes that resemble markers for the six different clusters. Depicted are genes with a p-value < 0.01 (Wilcoxon rank sum test) after bonferroni correction, and an average absolute fold-change > log2(1.3). Shown are z-scores of the average expression. d) UMAP representation of the expression levels of selected signature genes for activated/differentiated B cells. e) UMAP representation of analyzed cells from one healthy control and two ICU patients, representing early (first week, patient #1) and late (>7days, patient #5) phase after ICU admission. f) Percentage of cells belonging to a defined cluster among all sequenced B cells per donor. Each dot represents one time point from one single donor. Donors were grouped as “HC” for healthy controls (n = 3), “1st week” for patients within 7 days after ICU admission (n = 6) and “Late” for patients who have been admitted to the ICU for more than a week at the time of analysis (n = 8). The line indicates the median. Significance was determined by using a two-sided analysis of varience (ANOVA) folowed by Tukey’s multiple comparison test with corrected p values as indicated in the figure.

Article Snippet: Fluorescence-coupled antibodies used: IgA2-PE (Clone REA995, Miltenyi, Cat. No.130-117-763, dilution 1:50); CD27-PE (Clone REA499, Miltenyi, Cat. No. 130-114-166, dilution 1:50); CD38-PE (Clone IB6, Miltenyi, Cat. No. 130-113-427, dilution 1:50) and IgA-PE (Clone IS11–8E10, Miltenyi, Cat. No. 130-114-002, dilution 1:50).

Techniques: Isolation, Sequencing, MANN-WHITNEY, Expressing, Activation Assay, Control, Comparison

Journal: medRxiv

Article Title: In severe COVID-19, SARS-CoV-2 induces a chronic, TGF-β-dominated adaptive immune response

doi: 10.1101/2020.09.04.20188169

Figure Lengend Snippet: a) 5-marker MELC panel of SARS-CoV2-positive and control lungs (SARS-CoV2-negative). Respective patient characteristics are given in supplementary Fig. 4a. Each image of each patient depicts the same field of view of the same section, sequentially stained with the fluorescence-labelled antibodies indicated and the nuclear stain DAPI. Magenta arrows indicate IgA2+IgA+CD27 + CD38 + cells (containing a nucleus). Images contain 2048 × 2048 pixels and are generated using an inverted wide-field fluorescence microscope with a 20x objective, a lateral resolution of 325 nm and an axial resolution above 5 µm. Scale bar: 100 µm. b) Absolute numbers of IgA2+IgA+CD27 + CD38 + cells per field of view in all MELC runs acquired (two runs per patient except for COVID-19_A with four runs and Control_B with one single run; see and supplementary Fig. 4b). Each field of view is represented by a circle. The line indicates the median. Unpaired two sided Mann-Whitney U test. c) Region of interest of one exemplary control and COVID-19 lung (as in a)), showing an overlay of the indicated markers. White arrows point out IgA2+CD27 + CD38 + cells. Scale bar: 20 µm. (d-e) Bronchoalveolar lavage (BAL) cells for single cell sequencing were enriched for CD45 + cells via MACS and live cells were further sorted using FACS (see supplementary Fig. 4c). d) UMAP of 5459 cells representing clusters containing T cells, B cells and CD14 -expressing cells from patient #1 on day 59 following ICU admission. B and T cell cluster identification based on BCR/TCR, CD19, CD3E, CD4 and CD8A expression (see supplementary Fig. 4d). UMAP representation of expression of TGFB1, IL21, CD40LG and IFNG . e) UMAP coordinates and clustering was computed for 433 and 2723 cells from patient #9 on days 31 and 46 following ICU admission, respectively. UMAP representation of expression of TGFB1, IL21, CD40LG and IFNG at the two different time points is shown side by side.

Article Snippet: Fluorescence-coupled antibodies used: IgA2-PE (Clone REA995, Miltenyi, Cat. No.130-117-763, dilution 1:50); CD27-PE (Clone REA499, Miltenyi, Cat. No. 130-114-166, dilution 1:50); CD38-PE (Clone IB6, Miltenyi, Cat. No. 130-113-427, dilution 1:50) and IgA-PE (Clone IS11–8E10, Miltenyi, Cat. No. 130-114-002, dilution 1:50).

Techniques: Marker, Control, Staining, Fluorescence, Generated, Microscopy, MANN-WHITNEY, Sequencing, Expressing