Fig. 2

Phosphorylation of Jun proteins during the finfold and fin regenerations. (A) Comparison of amino acid sequences around the phosphorylation target site (Kallunki et al., 1996) of the human cJUN (GenBank NP_002219) with the corresponding regions of Junbs from human (GenBank CAG33122), mouse (AAA74916), and zebrafish (Junb, NM_213556; Junbl, NM_212750). (B) Anti-pJun antibody staining of regenerating larval finfold. Staining was not seen in the uncut finfold, but was detected (arrows) from 1 mpa to stages later than 24 hpa. Note that the signal was limited in cells close to the amputation plane within the domains of mRNA expression (see Figs. 1D–F). At 24 hpa, the signal was localized in the mesenchymal cells corresponding to the blastema cells expressing the junbl gene. (C, D) Anti-pJun antibody staining during adult fin regeneration at 6 hpa (C) and 2 dpa (D). At 6 hpa, the signal was observed in the epidermal cells; however at 2 dpa, the signal was localized in the growing blastema cells. Arrowheads in (D) mark the site of amputation. (E) Detection of the V5-tagged zebrafish c-Jun, Junb and Junbl proteins expressed in HEK293T cultured cells with anti-pJun antibody. The weak fluorescent signal for anti-pJun in the untransfected cells (top row) reflects the endogenous human JUN proteins. The expressed fish c-Jun, Junb and Junbl proteins were reactive with the anti-pJun antibody. (F) Western blot analysis of expressed zebrafish Jun proteins. The lower band in the right side panel (solid arrowhead) represents the endogenous human Jun proteins. The open arrowhead indicates the zebrafish Jun proteins that reacted with anti-V5 and anti-pJun antibodies. The scale bar represents 100 μm in (B) and (C, left panel), and 30 μm in (C, right panel).