Fig. 1.

Fig. 1.

ICER protein expression in zebrafish tissues. (A) Western blot analysis of protein expression in melanomas from the indicated zebrafish strains. Tissue samples were collected from skin and tumor tissues as shown. Western blotting for endogenous ICER expression was performed from whole tissue extracts using anti-ICER polyclonal antibodies (Cirinelli et al., 2022). The same membrane was stripped and re-probed with anti-α-Tubulin antibodies as a loading control (bottom panel). The left margin indicates the relative mobility of low range molecular weight markers from Bio-Rad (in kDa). The right margin indicates the relative mobility of ICER and α-Tubulin. Asterisks denote post-translationally modified forms of ICER (Mémin et al., 2011; Cirinelli et al., 2022). (B) Western blot analysis of FACS-sorted mitfa⁺ skin cells isolated from mitfa:mCherry⁺; crestin:EGFP⁺; tyrosinase⁻/⁻ zebrafish at 6 and 14 months post fertilization. Skin tissue was manually dissected, enzymatically dissociated, and filtered to generate a single-cell suspension. Live mitfa⁺ cells (DRAQ7⁻, mCherry⁺) were isolated via FACS. These mitfa⁺ skin cells include a mixed population primarily consisting of melanocytes and xanthophores. A total of 98,000 sorted cells pooled from 5–7 zebrafish were lysed in RIPA buffer supplemented with protease inhibitors. Lysates were clarified and subjected to western blotting as described in (A). ICER protein was not reduced in the tumor derived cells. While the absence of detectable protein may reflect post-transcriptional instability or proteasomal degradation of ICER, the possibility of transcriptional downregulation cannot be excluded, as mRNA levels were not assessed in this experiment.