Fig. 2

Letm1 localizes exclusively in mitochondria and displays a diel rhythm. (A) Schematic representations of letm1 gene and protein below with the locus (yellow box) of the deletion induced by the TALEN pairs. The region deleted (Δ16) is represented in “–.” The red asterisk represents a stop codon. TM, transmembrane region. 14-3-3, also known under the name of LETM Domain or LETM1 RBD (ribosomal binding domain) CC, coil–coil; EF, putative Ca²⁺-binding EF hand motif. Green bar indicates the protein fragment used to generate the Dre-Letm1 antibody. (B) Immunoblot using 6 day post fertilization zebrafish protein lysates from WT and letm1−/− with β-actin as a loading control. (C, D, E, F, G, H) Confocal images of WT and letm1−/− zebrafish cells immunolabeled with zebrafish anti-Letm1 (green) and stained with MitoTracker Red (magenta) and DAPI (blue). Scale bar 10 μm. (I) Representative immunoblots using 6 and 7 day post fertilization zebrafish protein lysates from WT and letm1−/− fish probed with anti-Letm1, anti-Tom20 and anti–β-actin. (J) Quantitative graph of four biological replicates (see Fig S1A–D) representing the relative protein level of Letm1 to β-actin over circadian time points. One-way ANOVA, n = 4.