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. 2018 Oct 30;9:4511. doi: 10.1038/s41467-018-06990-9

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — Phosphorylation of MPP9 by TTBK2 promotes UPS-mediated degradation of MPP9. a Immunoblots of lysates from hTERT RPE-1 cells after serum starvation. Tubulin was used as a loading control. b Immunoblots following immunoprecipitation (IP) with an anti-Flag antibody using lysates from hTERT RPE-1 cells overexpressing the indicated proteins. c Immunoblots of MPP9 after serum starvation in hTERT RPE-1 cells. Tubulin was used as a loading control. Relative amounts of MPP9 were quantified and normalized to tubulin. d Immunostaining of MPP9 (green) and ODF2-HA (red) in hTERT RPE-1 cells. Arrows, the distal ends of the mother centrioles. e Quantification of the fluorescence intensity of MPP9 at the distal ends of the mother centrioles from d (n = 40 cells for each group). f Immunoblots of MPP9 and S629-phosphorylated MPP9 in cycling hTERT RPE-1 cells. g Immunoblots following immunoprecipitation with an anti-MPP9 antibody using lysates from control- or TTBK2-siRNA-treated hTERT RPE-1 cells. h Immunoblots of TTBK2 and MPP9 after serum starvation in control- or TTBK2-siRNA-treated hTERT RPE-1 cells. Tubulin was used as a loading control. i Immunoblots of lysates from HEK293T cells co-overexpressing the indicated proteins. Tubulin was used as a loading control. Relative amounts of Flag-tagged MPP9 were quantified and normalized to tubulin. WT, wild-type; KD, kinase-dead. j Immunoblots of lysates from HEK293T cells co-overexpressing the indicated proteins. Tubulin was used as a loading control. Relative amounts of Flag-MPP9 were quantified and normalized to tubulin. k Immunoblots following immunoprecipitation with an anti-Flag antibody using lysates from HEK293T cells overexpressing the indicated proteins. l Mass spectrometry analysis showing the ubiquitination of MPP9 at K632 (blue). Yellow, phosphorylation sites. m Immunoblots of lysates from HEK293T cells co-overexpressing Flag-tagged MPP9-WT or the K632R mutant with TTBK2-GFP-WT or TTBK2-GFP-KD. Tubulin was used as a loading control. Relative amounts of Flag-MPP9 were quantified and normalized to tubulin. n Immunoblots following immunoprecipitation with an anti-Flag antibody using lysates from HEK293T cells overexpressing the indicated proteins. For e, bars represent the means ± S.E.M for three independent experiments. n.s., not significant, **p < 0.01, as determined by one-way ANOVA analysis. Scale bar: 1 μm (d)