Journal: STAR Protocols

Article Title: Protocol for efficient CRISPR-Cas9-mediated fluorescent tag knockin in hard-to-transfect erythroid cell lines

doi: 10.1016/j.xpro.2024.103016

Figure Lengend Snippet:

Article Snippet: Goat anti-mouse (IR700) , Advansta , Cat# R-05055-250.

Techniques: Virus, Recombinant, Membrane, Clone Assay, Ligation, Transfection, DNA Extraction, Isolation, SYBR Green Assay, Plasmid Preparation, Software, Cell Culture, Cell Counting

Journal: Frontiers in Immunology

Article Title: Negative Regulation of Interferon-β Production by Alternative Splicing of Tumor Necrosis Factor Receptor-Associated Factor 3 in Ducks

doi: 10.3389/fimmu.2018.00409

Figure Lengend Snippet: duMAVS or duTRIF colocalizes with duck TRAF3 (duTRAF3) or TRAF domain of duTRAF3 in the cytoplasm, but not spliced isoform of duTRAF3 (duTRAf3-S). (A) Duck embryo fibroblasts (DEFs) seeded in 12-well plates were transiently transfected individually with expression plasmids for HA-duTRAF3 or HA-duTRAF3-S for 24 h, and cells were then fixed and stained with antibody against the HA tag. Subsequently, cells were treated with FITC-conjugated goat anti-mouse secondary antibody and with 4′,6-diamidino-2-phenylindole (DAPI). (B) DEFs were transfected with Flag-duMAVS or Flag-duTRIF and HA-duTRAF3 or HA-duTRAF3-S. (C) DEFs transiently transfected with HA-T3319 and Flag-duMAVS or Flag-duTRIF. (D) DEFs transiently transfected with HA-duTRAF3-S and Flag-duTRAF3. Twenty-four hours after transfection, cells were fixed with 4% paraformaldehyde and stained with anti-HA or anti-Flag antibody. Secondary antibodies conjugated to Cy3 or FITC were used to visualize the stained Flag-tagged and HA-tagged proteins, respectively. DAPI show the nuclei of cells. Fluorescence was examined with a Zeiss LSM 510 Meta confocal microscope.

Article Snippet: The membranes were blocked in 1% bovine serum albumin (BSA) in TBST buffer for 1 h at room temperature and probed with indicated primary Abs for 1 h. After hybridizing with either goat anti-rabbit or goat anti-mouse secondary Abs, the membranes were visualized with ECL reagents (Advansta).

Techniques: Transfection, Expressing, Staining, Fluorescence, Microscopy