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Fig. 4 The esrom gene. (A) Predicted domains in Esrom. See text for full description. mo represents target sites for morpholinos. The positions of three mutations are indicated above the protein. (B) Esrom is phylogenetically closer to human PAM than Drosophila HIW and C. elegans RPM-1. (C) E3 ligase activity of 45 kDa Esrom C terminus containing the RING finger. Lanes 1-3 are controls. Lane 4 shows protein polyubiquitination in the presence of E1, E2 (UbcH5b) and Ub. (D,E) In situ hybridization with 30-hour-old zebrafish, shown here in lateral view. The antisense probe to esrom shows widespread label, with signal visible in all regions of the brain (D); no signal was detected with a sense probe (E). (F,G) Three-dimensional reconstructions of the eye of 48-hour-old fish labeled with antisense (F) or sense (G) probes to esrom, showing a lateral view. The gene is expressed uniformly in the developing retina. Lens labeling is nonspecific. (H,I) Western blot using a C terminus specific anti-PAM antibody. The antibody recognizes the 67 kDa C terminus end of Esrom (H), which is 98% identical to PAM, and a single 500 kDa protein from zebrafish embryo extract (I). (J-M) Esrom localization in retinal cells in vitro. (J,K) Label is detectable in the cytoplasm (arrowhead) and entire axon (arrow). Puncta are visible at high magnification (M, arrowhead). (J,L) Phase images; (K,M) the corresponding fluorescence images. (N-Q) Pre-absorption control; both axons were imaged at identical settings on a confocal microscope. (N,O) Without pre-absorption, the entire axon was labeled. (P,Q) With pre-absorption, very weak label was detected. Scale bar: 5 µm in L,M; 10 µm in J,K,N-Q; 100 µm in D-G; l, lens