mcherry mouse mab  (Elabscience Biotechnology)

Journal: Science Advances

Article Title: Trans-splicing facilitated by RNA pairing greatly expands sDscam isoform diversity but not homophilic binding specificity

doi: 10.1126/sciadv.abn9458

Figure Lengend Snippet: ( A ) Schematic diagrams of minigene constructs used to assess the effects of RNA secondary structure on trans-splicing of sDscam β 4 . ( B ) Predicted competing RNA pairings. Mutations introduced into dsRNA are indicated on the upper or lower mutated sequences (M2, M3, M4). Stem I has been validated in . Green arrows depict the activation of trans-splicing between different sDscam βs. ( C ) Validation of the effects of RNA pairings on trans-splicing by disruptive single mutations (M2, M3, M4) and compensatory double mutations (M24: M2 + M4; M34: M3 + M4). Data are expressed as means ± SD from three independent experiments. ( D ) Validation of trans-splicing at the protein level. The CDS of EGFP was split into two halves (EG and FP), followed by intronic sequences of sDscam β. EG: exon EG with 1 to 154 nt of intron 4 of the β1V13 cassette; FP: exon FG with 272 to 892 nt of intron 4 of β2. ( E ) Fluorescent photos of S2 cells transfected with WT and mutant plasmids containing CDS of EG/FP fused with intronic sequences from the β1V13 cassette and β2 (scale bars, 100 μm). ( F ) Detection of trans-spliced products by Western blotting using anti-EGFP antibody.

Article Snippet: Western blotting primary antibodies were used for relative quantification of sDscam: anti-mCherry tag mouse monoclonal antibody (1:5000; EarthOx, catalog no. E022110-01, RRID:AB_2687920) and anti–β-actin mouse monoclonal antibody (1:5000; Abcam, catalog no. ab8224, RRID:AB_449644).

Techniques: Construct, Activation Assay, Transfection, Mutagenesis, Western Blot

Journal: Science Advances

Article Title: Trans-splicing facilitated by RNA pairing greatly expands sDscam isoform diversity but not homophilic binding specificity

doi: 10.1126/sciadv.abn9458

Figure Lengend Snippet: See also fig. S9. ( A ) Schematic diagram of cell aggregation experiments. The mCherry-tagged sDscamβ proteins were expressed in Sf9 cells to test their ability to form cell aggregates. ( B ) Schematic diagram of the combination between the 5′ variable region and 3′ constant regions of sDscam β to form cis- and trans-spliced isoforms. The results of homophilic binding are summarized on the right-hand side. * indicates the lack of the 5′ variable region. Cis, cis-spliced isoforms; Trans, trans-spliced isoforms. ( C ) Homophilic binding of 64 cis- and trans-sDscamβ isoforms. Data quantitation of representative isoforms is shown. Data are expressed as means ± SD from three independent experiments. These data indicate that constant domains influence homophilic trans-binding ability. See also fig. S9. ( D ) A series of N-terminal truncations of the extracellular domain of sDscamβ fused to mCherry were subjected to cell aggregation assays. All sDscam truncations lacking the N-terminal Ig1 domain failed to form cell aggregates. ( E ) A series of domain deletion truncations were performed starting from the membrane-proximal FNIII3 domain. These data indicate that homophilic trans-binding is associated with constant extracellular domains of sDscamβ (scale bars, 100 μm).

Article Snippet: Western blotting primary antibodies were used for relative quantification of sDscam: anti-mCherry tag mouse monoclonal antibody (1:5000; EarthOx, catalog no. E022110-01, RRID:AB_2687920) and anti–β-actin mouse monoclonal antibody (1:5000; Abcam, catalog no. ab8224, RRID:AB_449644).

Techniques: Binding Assay, Quantitation Assay