Figure 4.
The role of Ref(2)P in Gox localization and ubiquitination. (A) Ref(2)P is specifically enriched in the shaft region of olf hair cells, which is labeled by HA–Gox. Maxillary palp 42 h APF. (B) Subcortical localization of Ref(2) in control and gox mutant olf hair cells, and their quantification. Line intensity profiles were obtained from 5–7 individual hairs per genotype using the PlotProfile function in Fiji and averaged after normalization with custom Python scripts. Gray line: Ref(2)P distribution in control; magenta line: Ref(2)P in gox KO. Error shading represents mean ± SD. (C) Subcortical Gox localization was lost in Ref(2)P RNAi. Right graph: quantification. Right panel: quantification of fluorescence intensity profiles. Gray line: HA–Gox in control; magenta line: HA–Gox in Ref(2)P knockdown cells. No formal statistical test was applied because the plots represent averaged fluorescence intensity distributions (D) Interaction of Gox2 with Ref(2)P and ubiquitination in S2 cells. Gox–Flag and Ref(2)P-Myc were co-expressed and immunoprecipitated with the tag antibodies. Immunoprecipitate of Ref(2)P containing a low molecular weight form of Gox (*, lane 4). The increased amount of high molecular weight forms of Gox was induced by Ref(2)P (compare lane 7 and 8). Note that cross-reactivity of HRP-conjugated antibodies to the IgG (marked with **) used for immunoprecipitation. (E) Properties of Gox8KR. Gox8KR (*) and Ref(2)P (**) were co-immunoprecipitated with each other (lane 8 and 12). *** indicates the band of Ref(2). Although Ref(2) caused a slight increase in Gox amount and molecular mass (lane 16), no specific increase in ubiquitination level was observed (lane 20). (F) WT gox and gox8KR were expressed by neur-Gal4. (G) Line intensity profiles showing the distribution of Flag (pink) and ER marker tdTomato–Sec61b (gray) in olf hair cells expressing UAS–Gox–Flag (top) or UAS–Gox8KR–Flag (bottom). Profiles were obtained and averaged as in B and C. No formal statistical test was applied; error shading represents mean ± SD. (H) AlphaFold 2 model of Gox/Osi23 (pLFFT score 68.12), and the location of the two mapped ubiquitinated lysine (K101 and K131) identified by the LC-MS/MS. Alpha helices are labeled (H1–H6). Molecular weight markers (kDa) are indicated for all blots, and uncropped images are provided in SourceData FS4.pdf.

The role of Ref(2)P in Gox localization and ubiquitination. (A) Ref(2)P is specifically enriched in the shaft region of olf hair cells, which is labeled by HA–Gox. Maxillary palp 42 h APF. (B) Subcortical localization of Ref(2) in control and gox mutant olf hair cells, and their quantification. Line intensity profiles were obtained from 5–7 individual hairs per genotype using the PlotProfile function in Fiji and averaged after normalization with custom Python scripts. Gray line: Ref(2)P distribution in control; magenta line: Ref(2)P in gox KO. Error shading represents mean ± SD. (C) Subcortical Gox localization was lost in Ref(2)P RNAi. Right graph: quantification. Right panel: quantification of fluorescence intensity profiles. Gray line: HA–Gox in control; magenta line: HA–Gox in Ref(2)P knockdown cells. No formal statistical test was applied because the plots represent averaged fluorescence intensity distributions (D) Interaction of Gox2 with Ref(2)P and ubiquitination in S2 cells. Gox–Flag and Ref(2)P-Myc were co-expressed and immunoprecipitated with the tag antibodies. Immunoprecipitate of Ref(2)P containing a low molecular weight form of Gox (*, lane 4). The increased amount of high molecular weight forms of Gox was induced by Ref(2)P (compare lane 7 and 8). Note that cross-reactivity of HRP-conjugated antibodies to the IgG (marked with **) used for immunoprecipitation. (E) Properties of Gox8KR. Gox8KR (*) and Ref(2)P (**) were co-immunoprecipitated with each other (lane 8 and 12). *** indicates the band of Ref(2). Although Ref(2) caused a slight increase in Gox amount and molecular mass (lane 16), no specific increase in ubiquitination level was observed (lane 20). (F) WT gox and gox8KR were expressed by neur-Gal4. (G) Line intensity profiles showing the distribution of Flag (pink) and ER marker tdTomato–Sec61b (gray) in olf hair cells expressing UAS–Gox–Flag (top) or UAS–Gox8KR–Flag (bottom). Profiles were obtained and averaged as in B and C. No formal statistical test was applied; error shading represents mean ± SD. (H) AlphaFold 2 model of Gox/Osi23 (pLFFT score 68.12), and the location of the two mapped ubiquitinated lysine (K101 and K131) identified by the LC-MS/MS. Alpha helices are labeled (H1–H6). Molecular weight markers (kDa) are indicated for all blots, and uncropped images are provided in SourceData FS4.pdf.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal