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Fig. 7 Mak kinase activity is required for Mak protein localization in the ciliary axoneme, axoneme formation and photoreceptor maintenance. (A) A scheme of generation of three types of Mak kinase-dead mutants: AR Mak, ADF Mak and DM Mak. Zebrafish Mak has a catalytic domain, which contains an ATP-binding motif, KR, and an autophosphorylation site, TDY. AR Mak and ADF Mak were generated by conversion of KR and TDY into AR and ADF, respectively. DM Mak has both the AR and ADF mutations. (B) In vitro kinase assay of Mak using a substrate, MBP. Only wild-type Mak phosphorylated MBP. The number of blots is shown in Table S3. (C) Labeling of 6-dpf wild-type ONL carrying the transgene Tg[crx:eGFP], Tg[crx:eGFP-WT MAK], Tg[crx:eGFP-AR MAK], Tg[crx:eGFP-ADF MAK] or Tg[crx:eGFP-DM MAK] with anti-GFP antibody (green). (D) Labeling of 6-dpf wild-type and pday mutant retinas carrying the transgene Tg[crx:eGFP], Tg[crx:eGFP-WT MAK], Tg[crx:eGFP-AR MAK], Tg[crx:eGFP-ADF MAK] or Tg[crx:eGFP-DM MAK] with zpr1 (red) and anti-GFP (green) antibodies. zpr1-positive photoreceptors were degenerated in dorso-central retinas of pday mutants carrying Tg[crx:eGFP] (white dotted line). However, photoreceptor degeneration was rescued only in pday mutants carrying Tg[crx:eGFP-WT MAK], but not in pday mutants carrying kinase-dead mutant transgenes (white dotted line). Images of wild-type and pday mutant retinas carrying the transgenes Tg[crx:eGFP] and Tg[crx:eGFP-WT MAK] are serial sections of the same retinas shown in Fig. 4A. (E) Labeling of 3-dpf wild-type and pday mutant ONL carrying the transgene Tg[crx:eGFP], Tg[crx:eGFP-WT MAK], Tg[crx:eGFP-AR MAK], Tg[crx:eGFP-ADF MAK] or Tg[crx:eGFP-DM MAK] with anti-acetylated α-tubulin antibody (red). Axoneme formation defects were rescued only in pday mutants carrying Tg[crx:eGFP-WT MAK] (arrowheads), but not in pday mutants carrying kinase-dead mutant transgenes (asterisks). In C-E, nuclei were counterstained with Hoechst 33342 (blue). Sample sizes are shown in Table S3. Scale bars: 5 µm (C,E); 40 µm (D).