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Fig. 5 During zebrafish development, Rps10/eS10 ubiquitination is crucial for establishing the ribosome ubiquitination pattern. (A) Alignment of WT and K139/140R rps10 sequences. The sequencing chromatogram of the rps10 K139/140R embryos is indicated below. (B,C) Detection of Rps10/eS10 ubiquitination with or without Znf598 overexpression in rpl36-FLAG and rps10 K139/140R;rpl36-FLAG embryos. Total lysates were subjected to immunoblotting analysis of Znf598-Myc and Tubulin (B). FLAG-IPs were subjected to immunoblotting analysis with an anti-Rps10 antibody (C, left and middle) and protein staining (C, right). White and black arrowheads indicate nonubiquitinated or ubiquitinated Rps10/eS10 signals, respectively. Asterisks indicate nonspecific signals. (D) Comparison of ribosome ubiquitination pattern in rpl36-FLAG, rps10 K139/140R;rpl36-FLAG, and MZznf598;rpl36-FLAG embryos. FLAG-IPs were subjected to immunoblotting analysis with an anti-Ubiquitin antibody (left and middle) and protein staining (right). Black arrowheads indicate putative ubiquitinated Rps10/eS10 signals. White arrowheads indicate reduced ubiquitination signals in rps10 K139/140R;rpl36-FLAG and MZznf598;rpl36-FLAG embryos.