Journal: The Journal of Biological Chemistry

Article Title: Redirecting the specificity of tripartite motif containing-21 scaffolds using a novel discovery and design approach

doi: 10.1016/j.jbc.2023.105381

Figure Lengend Snippet: Fluorescence microscopy and plate reader analysis of MALT1 and EED degradation. HEK 293F cells were cotransfected with a plasmid encoding MALT1-tGFP ( A and B ) or EED-tGFP ( C and D ) as well as a plasmid encoding an mCherry tag fused to the indicated TRIM21–Tn3 variant, separated by a T2A peptide in order to decouple degradation from expression. A and C , after 72 h, 20× images were acquired with a Zeiss Axio Observer with AxioVision 5 software, and composite images were generated with ImageJ. Images are representative of biological triplicates. Scale bar represents 100 μm. B and D , mean tGFP (488 ± 9 nm excitation; 510 ± 9 nm emission) and mCherry fluorescence (580 ± 20 nm excitation; 630 ± 20 nm emission) were also measured after 72 h using a BioTek SynergyMX plate reader (BioTek Instruments, Inc) that analyzed a 3 × 3 grid distributed across the well surface area. Background-subtracted tGFP fluorescence of each well was normalized by the mCherry fluorescence and divided by the mean mCherry-normalized tGFP fluorescence of the appropriate control sample. Data represent mean ± SD of biological triplicates. Statistical significance was determined by a two-tailed unpaired Student’s t test, and statistical data are shown in Table S4 . ∗∗∗ p ≤ 0.001, and ∗∗∗∗ p ≤ 0.0001. EED, embryonic ectoderm development protein; HEK, human embryonic kidney cell line; MALT1, mucosa-associated lymphoid tissue lymphoma translocation protein 1; tGFP, TurboGFP; Tn3, third fibronectin type III domain of human tenascin-C; TRIM21, tripartite motif containing-21.

Article Snippet: After 72 h, 20× images were acquired with a Zeiss Axio Observer with AxioVision 5 software (Zeiss United States), and composite images were generated with ImageJ ( ).

Techniques: Fluorescence, Microscopy, Plasmid Preparation, Variant Assay, Expressing, Software, Generated, Two Tailed Test, Translocation Assay