(A) Sequence trace analysis of the mutated codon in homozygous (blt/blt) compared to heterogygous (blt/+) mice, showing an Arg-to-Trp substitution at position 1092. (B) Linear schematic of the 13 C2H2 zinc finger (ZF) domains (shaded boxes) in Zfp335. Asterisk indicates the R1092W bloto mutation in ZF12 (black box). Diagram drawn to approximate scale. (C) Multiple sequence alignment of predicted Zfp335 orthologs from dog (Canis lupus), pig (Sus scrofa), human (Homo sapiens), mouse (Mus musculus), chicken (Gallus gallus), and zebrafish (Danio rerio). Asterisk indicates Arg residue affected by bloto mutation. Amino acids are colored according to their physicochemical properties. (D) Quantitative RT-PCR analysis of Zfp335 mRNA from indicated FACS-purified thymocyte subsets and naïve T cells (n = 3–4 mice, mean ± s.d. for biological replicates); ISP, immature CD8+ thymocytes identified by tlack of TCRβ expression; results are presented relative to expression of Hprt. (E) Western blot for Zfp335 protein in the thymocytes from wild-type (+/+) and homozygous mutant (b/b) mice, with actin as loading control. (F) Immunofluorescence analysis of Zfp335 nuclear localization in mature CD4SP thymocytes; nucleus counterstained with DAPI. (right) Secondary antibody-only negative staining control. Scale bar: 2 μm. (G) Frequency of CD4+ and CD8+ T cells differentiating from blt/blt hematopoietic stem cells transduced (Thy1.1+) with either wild-type Zfp335 (Zfp335WT) or control MSCV-IRES-Thy1.1 vector, compared to non-transduced (Thy1.1−) cells from the same mouse, 8 to 10 weeks after reconstitution of irradiated hosts. (H) Transduced (Thy1.1+) cells as a percentage of indicated thymocyte and T cell subsets from irradiation chimeras that had received bone marrow retrovirally transduced with WT Zfp335, bloto Zfp335 or control vector. Data points are connected by a separate line for individual mice. Data are representative of three independent experiments.