Fig 1: Dynamic regulation of UGA/Sec recoding during myoblast differentiation.a Constructs of the FLAG-SELENON expression vector and its mutants ?SECIS and GGA. TM (transmembrane domain), EF-hand domain, and Sec residue in the catalytic domain are indicated. b Expression of FLAG-SELENON and its mutants in HEK293T cells, detected by western blotting with an anti-FLAG antibody. This experiment was done once. c Northern blotting of cytoplasmic tRNASec in total RNA obtained on each day of differentiation. Relative steady-state level of tRNASec on each day of differentiation compared with the level on Day 0 is shown below. Results were normalized relative to the band intensity of 5.8 S rRNA in the same sample. Data are presented as mean values ±S.D. Statistical significance was determined by unpaired two-tailed t test; n = 3 biologically independent samples. d Volcano plot of RNA-seq transcriptome data showing altered gene expression [log2(fold-change)], with p values, on Day 5 versus Day 0 of Hu5/KD3 cell differentiation. UGA/Sec recoding factors are indicated, and significantly differentially expressed genes (two-sided p value <0.05 determined by likelihood ratio test) are highlighted in red. e Expression analyses of GAPDH (control) and SEPSECS in differentiating Hu5/KD3 cells by western blotting on the indicated days. The expression level of SEPSECS was normalized to that of GAPDH, making the relative expression level on Day 0 = 1.00 (shown below). The samples derived from the same experiment and the gels/blots were processed in parallel. This experiment was repeated more than three times independently with similar results. f Subcellular localization of SEPSECS on the indicated days in differentiating Hu5/KD3 cells immunostained with an anti-SEPSECS antibody (green). Nuclei were stained with DAPI (blue) and myotubes were stained with an anti-myosin antibody (red). Magnification: ×100. Exposure time: 150 ms (Day 0) or 300 ms (Day 3 and 5). This experiment was done once. Source data and unprocessed gel image are provided in Source Data file.