Journal: Molecular Cell

Article Title: Rapid characterization of spike variants via mammalian cell surface display

doi: 10.1016/j.molcel.2021.11.024

Figure Lengend Snippet:

Article Snippet: All images were collected with a Nikon Ts2R-FL inverted research microscope equipped with a CF160 Plan Apochromat Lambda 60x oil immersion objective lens.

Techniques: Control, Recombinant, Expressing, Transfection, Staining, Blocking Assay, Protease Inhibitor, Plasmid Preparation, DNA Purification, Luciferase, Cloning, Software, Binding Assay, Sterility, Magnetic Beads, Cell Culture, Imaging, Microscopy, Electron Microscopy

Journal: Nature Communications

Article Title: SDE2 integrates into the TIMELESS-TIPIN complex to protect stalled replication forks

doi: 10.1038/s41467-020-19162-5

Figure Lengend Snippet: a Human SDE2 domain structures and roles of the processed SDE2 Ct in the replication stress response and replication fork integrity. b Representative images of SDE2:EdU-biotin PLA foci with schematic of the PLA design. The biotin:biotin PLA was used as a positive control to mark replicating cells. M: mouse, R: rabbit, primary antibodies used. Scale bar, 10 μm. c Quantification of cells positive for SDE2:EdU PLA foci and the number of foci per nuclei. A representative graph from two independent experiments is shown. More than 150 cells were scored per experimental condition. d PLA between SDE2-GFP and endogenous PCNA. Scale bar, 10 μm. e Western blot (WB) analysis of iPOND samples from EdU pulse and thymidine chase to reveal the proteins specifically associated with the replisome. f Schematic of the SDE2-APEX2 in situ proximity biotinylation to tag proteins proximal to SDE2. g Fluorescence imaging of SDE2-APEX2 labeling of nuclear proteins, detected by streptavidin-conjugated Alexa Fluor (AF) 594. Nuclear localization of SDE2-GFP was confirmed by GFP fluorescence. Scale bar, 10 μm. h WB analysis of biotinylated proteins by proximity labeling. U2OS cells stably expressing SDE2-APEX2 were incubated with BP and H 2 O 2 , and biotinylated proteins were enriched by streptavidin pull-down.

Article Snippet: The beads were subsequently washed four times with NETN150 lysis buffer, and biotinylated proteins were eluted by boiling the beads in 2× Laemmli sample buffer for 5 min. For the imaging of SDE2-Flag-APEX2, U2OS cells on coverslips were treated with BP and H 2 O 2 , and fluorescence labeling was performed by incubating coverslips with Alexa Fluor 594-coupled streptavidin (Thermo Fisher) in 1% BSA, incubated in a humidifying chamber at 37 °C for 45 min. Coverslips were analyzed with the Eclipse Ts2R-FL inverted Nikon fluorescence microscope equipped with the Nikon DSQi2 digital camera.

Techniques: Positive Control, Western Blot, In Situ, Fluorescence, Imaging, Labeling, Stable Transfection, Expressing, Incubation