Extended Data Fig. 8: EXSISERS can be programmed with different reporters and effectors.
From: Non-invasive and high-throughput interrogation of exon-specific isoform expression
a,b, HaloTag was flanked by intein-CC domains and type I and type II transmembrane domains (TMDs), resulting in a surface functionalization with a HaloTag inserted into exon 10 of MAPT. c, Cells were incubated with chloroalkane-functionalized Alexa Fluor 660 (3.5 µM) for 10 min at 37 °C and washed with HBSS. d, Anti-pan-tau immunoblot showed the typical tau pattern indicating scarless excision. Data shown represent one independent experiment. e, Epifluorescence microscopy image of anti-pan-tau immunocytochemistry of EXSISERSMAPT:10Halo cells after MAPT induction showing filamentous tau staining. Data shown represent 2 independent experiments f, AF660 live-cell-staining after MAPT-induction resulted in a distinct membrane staining. Data shown represent 2 independent experiments. g, scAvidin was used as a high-affinity binding domain for biotinylated compounds. Test constructs for both designs contained mNG-NLS as surrogate exteins, which are expected to be spatially decoupled after the excision of the intein-domains. h,i, Surface localization of scAvidin or HaloTag in transfected HEK293T was confirmed by live-staining with biocytin-AF594 and chloroalkane-AF660, while mNeonGreen-NLS (mNG-NLS) was detected in the nucleus. Data shown represent one experiment from 2 cell culture wells. j, The binding moiety of the membrane anchors was exchanged for NLuc flanked by furin sites (FS), such that the Golgi-resident furin can cleave off NLuc and releases it into the supernatant. k, NLuc activity was measured over time in the supernatant of cells expressing the sheddable NLuc without/with furin sites. Data shown represent the mean values ± s.d. (n = 3 biological replicates). *** denotes p < 0.001 of selected Bonferroni MCT after two-way ANOVA with repeated measures (full statistical results in Supplementary Table 1). l, Intein-flanked mNG was inserted into the mouse Tubb3 gene. m, Immunoblot confirms successful intein splicing of mNG. Data shown represent 7 independent experiments. n, Mouse N2a cell line with insertion of the intein-mNG reporter into Tubb3 shows typical Tubb3 filaments via immunocytochemistry. The fluorescent mNG signal (inset) was observed throughout the cell, indicating successful post-translational splicing. Data shown represent 2 independent experiments. Unprocessed blots and numerical source data are provided in Source Data Extended Data Fig. 8.
