FIGURE 1.

Identification of two SATB1 protein isoforms in murine thymocytes. (A) SATB1 protein consists of the following domains and structural features: ULD–ubiquitin-like domain, CUTL–CUT-like domain, CUT1 and CUT2 domains, EP–the peptide encoded by the predicted extra exon of the long Satb1 isoform, Q–compositional bias represented by a poly-Q domain and a stretch of prolines, HD–homeodomain. In this study, we used two custom-made antibodies (Davids Biotechnology): an antibody targeting the first 330 amino acids of all isoforms (cannot discriminate between the two protein isoforms) and the long isoform antibody specifically targeting the extra peptide of the long isoform. (B) Comprehensive list of quantified Satb1 isoforms based on stranded-total-RNA-seq experiment in thymocytes. Data are the mean ± s.d. (C) Design of the confirmatory RT-qPCR experiment to quantitate relative expression of the short and long Satb1 isoforms in murine thymocytes. (D) RT-qPCR results for the relative mRNA levels of the two Satb1 isoforms. Values from 3 technical and 2 biological replicates were normalized to Dnmt1 mRNA levels. Data are the mean ± s.d. p values by Student’s T-test. (E) The custom-made antibody detecting the long SATB1 protein isoform cannot detect the short SATB1 isoform. Protein extracts (80 µg) prepared from transfected HEK293 cells with plasmids expressing GFP, GFP-SATB1-long and GFP-SATB1-short. Western blotting performed with antibodies detecting either the long SATB1 isoform, all SATB1 isoforms or GFP. (F) Scheme for the approach utilized to detect the SATB1 long isoform and validate the custom-made long isoform-specific antibody (Davids Biotechnology). Whole cell thymocyte protein extracts (sample 1) were prepared and incubated with a custom-made antibody against the long SATB1 isoform. The immunoprecipitated material (sample 2) was kept and the immunodepleted material was subjected on a second immunoprecipitation reaction utilizing an antibody detecting epitopes on both long and short SATB1 isoforms (Santa Cruz Biotechnology, sc-376096). The material from the second immunoprecipitation reaction was kept (sample 3). The second biological replicate is depicted in Supplementary Figures S1A, B; including the thymocyte extract immunodepleted for all SATB1 isoforms (sample 4). (G) Western blot analysis for the samples described in (F). The whole thymocyte protein extract (1), immunoprecipitated long SATB1 protein (2) and immunoprecipitated short SATB1 protein (3). UPPER PANEL: Western blot analysis utilizing a SATB1 antibody detecting only the long SATB1 isoform. LOWER PANEL: Western blot analysis utilizing a SATB1 antibody detecting all SATB1 isoforms. (H) Confocal and STED microscopy images indicating the subnuclear SATB1 cage-like pattern. The super-resolution microscopy unveils that the cage-like pattern is actually composed of individual, mostly round, 40–80 nm large speckles.