Fig. 4: Exon 15 of TBC1D23 regulates erythropoiesis in mice and zebrafish.
From: The functional landscape of alternative splicing in hematopoietic lineage commitment
a Schematic of the WT and E15-KO alleles, with arrows indicating primer sites and expected PCR product sizes (Top). Genotyping of E15-KO mice (Bottom). b RT-PCR validated the specific deletion of the exon 15-containing isoform (Tbc1d23-L) in E15- KO mice. c Immunoblot of Tbc1d23 total protein level in WT and E15-KO mice bone marrow. d Quantification of immunoblots in (c) (n = 3 independent experiments). e Peripheral blood RBC numbers, HCT%, and HGB in E15-KO mice (n = 28 mice) in comparison to WT (n = 34 mice). f Numbers of whole white cells (WBC), lymphocytes (LYM), monocytes (MON), and granulocytes (GRA) in E15-KO mice (n = 34 mice) in comparison to WT (n = 28 mice). g Schematic of morpholino oligonucleotide (MO) targeting Tbc1d23-E15. The horizontal line shows the position of Tbc1d23-E15 MO (E15-MO) (Top). RT-PCR validated the specific knockdown of Tbc1d23-L in control-MO-injected (Ctrl-MO) and E15-MO-injected (E15-MO) zebrafish embryos at 48 hpf (Bottom). h Immunoblot of total Tbc1d23 protein levels in zebrafish embryos following Ctrl-MO or E15-MO treatment. i Quantification of immunoblots in (h) (n = 3 independent experiments). j Fluorescence images of transgenic zebrafish Tg (Gata1: DsRed) in Ctrl-MO, and E15-MO zebrafish embryos at 48 hpf. k Quantification of the Gata1: DsRed intensity at the CHT region of Ctrl-MO and E15-MO (n = 20 zebrafish/group). l Representative images of O-dianisidine staining for hemoglobin in Ctrl-MO and E15-MO zebrafish embryos at 48 hpf. m Quantification of O-dianisidine intensity for embryos injected with Ctrl-MO (n = 34 zebrafish) and E15-MO (n = 26 zebrafish). For Statistical analysis, the following tests were used: Two-tailed Mann-Whitney U tests (e, f, k, m), Unpaired two-tailed Student’s t-test (d, i). Experiments of (a, b, g) were repeated three times, consistently yielding similar results in each iteration. Data in (d, i) are represented as mean  ± s.d and (e, f) are represented as mean  ± s.e.m, and pooled from three independent experiments. Source data are provided as a Source Data file.
