Journal: Molecular Cell

Article Title: Single-molecule analysis reveals cooperative stimulation of Rad51 filament nucleation and growth by mediator proteins

doi: 10.1016/j.molcel.2020.12.020

Figure Lengend Snippet:

Article Snippet: E. coli DH5alpha , NEB , Cat# C2987H.

Techniques: Recombinant, Lambda DNA Preparation, Purification, Expressing, Software, Confocal Microscopy

Journal: Microbiology Spectrum

Article Title: Duck Enteritis Virus Protein Kinase US3 Inhibits DNA Sensing Signaling by Phosphorylating Interferon Regulatory Factor 7

doi: 10.1128/spectrum.02299-22

Figure Lengend Snippet: DEV US3 phosphorylates IRF7 and blocks IRF7 dimerization and nuclear translocation. (A) IRF7-HA was cotransfected with empty vector or US3- or US3K213A-expressing plasmid into DEFs. The cells were lysed 36 h later and left untreated or treated with lambda PP prior to SDS-PAGE and Western blotting. (B) DEFs were transfected with empty vector or US3- or US3K213A-expressing plasmid. The cells were lysed 36 h later, and Western blotting was performed with anti-IRF7 antibodies to detect IRF7 phosphorylation. (C) DEFs were transfected with the indicated plasmids for 36 h before coimmunoprecipitation and immunoblot analysis with the indicated antibodies. (D) DEFs were transfected with US3 or US3K213A plasmid; 24 h later, cells were either left untreated or transfected with ISD for 12 h before confocal microscopy. Bars, 10 μm.

Article Snippet: To confirm that DEV US3 mediates the phosphorylation of IRF7, cell lysates were subjected to a dephosphorylation assay using lambda protein phosphatase (PP) (New England Biolabs) ( ).

Techniques: Translocation Assay, Plasmid Preparation, Expressing, SDS Page, Western Blot, Transfection, Confocal Microscopy

Journal:

Article Title: RPA is an initiation factor for human chromosomal DNA replication

doi:

Figure Lengend Snippet: RPA is recruited to DNA replication foci and becomes phosphorylated in vitro. (A) Comparison of bound RPA in G1 and S phase nuclei in vivo. Nuclear extracts (25 µg protein per lane) from mimosine- arrested late G1 phase and from early S phase cells were analysed by western blot using antibodies specific for MCM5 as a control and polyclonal antibody pAb-RPA1. (B) Nuclear binding and phosphorylation of RPA during initiation of DNA replication in vitro. G1 phase nuclei isolated from in vitro incubations done in replication buffer (lane 1), 100 µg S100 cytosolic extract (lane 2), 30 and 100 ng rhRPA (lanes 3 and 4) and 100 ng rhRPA plus 35 µg of fraction QB (lane 5). The samples shown in the right-hand panel were treated with λ phosphatase before loading onto the same gel. The Rpa70 and Rpa32 subunits were visualised with pAb-RPA1. Phosphorylated Rpa32 is indicated as pRpa32. (C) Confocal microscopy. G1 phase nuclei were incubated in buffer (top row), in 100 ng of rhRPA (second row), in 100 ng rhRPA supplemented with 35 µg QB (third row) and in 100 µg of unfractionated S100 (bottom row). High-resolution micrographs are shown of RPA foci (stained with pAb-RPA1, red), replication foci (dig-UTP, green) and merged images of the same set of nuclei.

Article Snippet: Phosphatase treatments were performed after the PBS wash by resuspending the nuclei in 20 µl of λ phosphatase buffer containing 2 mM MnCl 2 and incubating for 30 min at 30°C in the presence of 200 U λ phosphatase (NEB).

Techniques: In Vitro, In Vivo, Western Blot, Binding Assay, Isolation, Confocal Microscopy, Incubation, Staining

Journal: Cancer Research

Article Title: Protein Disulfide Isomerase Expression Is Related to the Invasive Properties of Malignant Glioma

doi: 10.1158/0008-5472.can-05-4589

Figure Lengend Snippet: Figure 5. Verification of PDI expression on the cell surface. A, flow cytometry: Relative fluorescence intensity of nonpermeabilized U373 cells incubated with PDI antibody and stained with FITC-conjugated secondary antibody compared with the control cells, not exposed to the PDI antibody. Inset, confocal microscopy of the same U373 cell, stained with PDI antibody. B, inhibition of U373 cell adhesion to the plastic surface after exposure to a PDI monoclonal antibody. The attached cells were counted after 2 and 24 hours. Five measurements in each group. Columns, mean; bars, SD.

Article Snippet: The secondary antibody used was isotope-specific goat anti-mouse conjugated to FITC (SouthernBiotech, Birmingham, AL).

Techniques: Expressing, Flow Cytometry, Fluorescence, Incubation, Staining, Control, Confocal Microscopy, Inhibition

Journal: Cancer Research

Article Title: Protein Disulfide Isomerase Expression Is Related to the Invasive Properties of Malignant Glioma

doi: 10.1158/0008-5472.can-05-4589

Figure Lengend Snippet: Figure 4. Localization of the PDI in glioblastoma samples obtained from low- and high-generation xenograft tumors and detection of PDI expression by invasive and migrating glioblastoma multiforme cells. A, photomicrographs of paraffin-embedded sections of xenograft tumor specimens immunostained for PDI with monoclonal mouse antibody and contrast stained with hematoxylin. Top, an invasive, low-generation, glioblastoma multiforme tumor in the rat brain; bottom, tumor xenograft displaying both angiogenic and invasive properties in the rat brain (high-generation tumor). There is some positive PDI staining in the invasive margin (black arrow) of the tumor, but the tumor core is PDI negative (white arrow). Bar, 100 Am. B, verification of differential expression of PDI by Western blots. Lanes 1 and 3, protein samples from low-generation tumors; lanes 2 and 4, tumor samples taken from the high generation. The samples were obtained from two different patients. Lanes 1 and 2, patient A; lanes 3 and 4, patient B. Different bands indicate different members of the PDI protein family, recognized by the same antibody. C, photomicrograph of a human glioblastoma multiforme biopsy, immunostained for PDI and contrast stained with hematoxylin. Insets, a high-power view of the tumor cells from different areas of the tumor: tumor core (top), invasive tumor rim (middle), and invading tumor cells (bottom) showing positive PDI staining in the invasive tumor rim. Bar, 500 Am. D, photomicrograph of migrating tumor cells from the U373 cell line spheroid, immunostained for PDI with FITC-conjugated secondary antibody. DAPI was used as nuclear stain. Magnifications, 10 (top left), 20 (right top), and 40 (right middle). Bottom, control staining of cell nuclei with DAPI, without primary antibody. Bar, 300 Am.

Article Snippet: The secondary antibody used was isotope-specific goat anti-mouse conjugated to FITC (SouthernBiotech, Birmingham, AL).

Techniques: Expressing, Staining, Quantitative Proteomics, Western Blot, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Soluble CD23 Controls IgE Synthesis and Homeostasis in Human B Cells i

doi: 10.4049/jimmunol.1102689

Figure Lengend Snippet: (A) Human tonsillar B cells were cultured for 8 days with IL-4 (200IU/ml) and anti-CD40 (1μg/ml) and stimulated for 30 minutes with media alone, derCD23 (3μM/48μg/ml) or triCD23 (1μM/84μg/ml). Cells were stained with mouse anti-CD21, plus secondary anti-mouse-Alexa 594 (red), anti-IgE-FITC (green), and the nuclear stain Hoescht (blue). Cells were visualized by confocal microscopy and images show a single field of view, with the bottom panel showing a three-color overlay. The white arrow indicates strong capping of mIgE with mCD21 (n=1, representative of 3). (B) Human tonsillar B cells were cultured for 12 days with IL-4/anti-CD40 alone, + anti-CD21 (0.1, 1 or 10μg/ml), + triCD23 (1μM) or + aCD21 (10μg/ml) & triCD23. IgE secretion was analysed by ELISA on day 12, relative to cells cultured with IL-4/anti-CD40 alone (n=2). (C) Cells were cultured as described in (A) and stimulated with anti-CD21 (10μg/ml), triCD23 (1μM) or anti-CD21 & triCD23. Cells were stained for mCD21 (red) and the nucleus (blue) and visualized by confocal microscopy (n=1).

Article Snippet: Goat anti-human IgE-FITC (1:500) (Vector) and monoclonal mouse anti-human IgG-APC (1:50) (Miltenyi Biotec) were added and incubated on ice in the dark for 45 minutes.

Techniques: Cell Culture, Staining, Confocal Microscopy, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: KDM4-dependent DNA breaks at active promoters facilitate +1 nucleosome eviction

doi: 10.1101/2023.07.14.548993

Figure Lengend Snippet: (A) Eviction of H2B-GFP and H3-GFP in nuclei treated with different concentrations of nickase. H2B-GFP and H3-GFP expressor HeLa nuclei treated with 0.95 M NaCl, or 10 µg/ml EBr / 0.75 M NaCl, respectively, after treatment with nickase used at the concentrations indicated in the figure. The curves refer to 200-2000 G1 phase cells gated according to their DNA content. Bars represent SEM. (B) Double-strand fragment size distribution of the DNA after nickase+S1 treatment. The nicks were converted to double-strand breaks by S1 nuclease and the ds fragments analyzed by CHEF as descibed in Methods and Methods. Lane 1: 225-2200 kb Pulse marker; lane 2: 1 kb DNA ladder; lane 3: lambda DNA; lane 4: untreated control; lane 5: 0.5 U/ml nickase; lane 6: 0.5 U/ml nickase + 1000 U/ml S1; lane 7: 1000 U/ml S1. (C) The fluorescent halo radius, determined by laser scanning cytometry, is plotted as a function of SG concentration. Upon addition of the dye the halo radius gradually increases as the negatively supercoiled plectonemic DNA loops progressively uncoil and become increasingly relaxed. Complete relaxation of the loops is observed between 1-1.7X SG concentration. Additional intercalation causes the relaxed DNA loops to form positive plectonemic writhes, thus decreasing the halo radius. (D-E) Nicks detected in nuclear halo samples of Jurkat cells by STED microscopy. Lamin B was detected by immunofluorescence (blue and green in the upper and lower images, respectively), DNA was stained by PI (green and blue in the upper and lower images respectively). Lamin B and nicks (red) only were imaged at superresolution. (F) Lamina-proximal TOP2β was detected on nuclear halo samples by confocal microscopy. Lamin B was detected by immunofluorescence (as before), TOP2β was detected by an antibody prepared in C. Austin’s lab and DNA was stained by PI (as before). The signal in the DNA channel of the upper images of Panels D-F were amplified to better visualize the nuclear halos. (G) Co-localization of lamina-proximal nicks and TOP2β in nulcear halo samples before and after etoposide treatment, visualized by confocal microscopy. TOP2β was detected using a rabbit polyclonal antibody (Santa Cruz: H-286; green)), DNA POL I labeled nicks were detected by anti-biotin antibody (red). Representative microscopic images are shown.

Article Snippet: Topoisomerases were stained using TOP2α or TOP2β specific antibodies (from Santa Cruz or C. A. Austin’s lab, at 1:100 dilution in 5 mg/ml BSA/PBS/EDTA, 4°C, overnight).

Techniques: Marker, Lambda DNA Preparation, Control, Cytometry, Concentration Assay, Microscopy, Immunofluorescence, Staining, Confocal Microscopy, Amplification, Labeling