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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. 5 — TTBK2 phosphorylates MPP9 mainly at S629 site. a Immunoblots of lysates from HEK293T cells co-overexpressing Flag-MPP9 and the indicated cilia-related kinase. Tubulin was used as a loading control. b Immunoblots of lysates from HEK293T cells co-overexpressing Flag-tagged MPP9 full-length (FL) or the indicated MPP9 truncation mutants with TTBK2-GFP-WT or TTBK2-GFP-KD. Tubulin was used as a loading control. WT, wild-type; KD, kinase-dead. c Immunoblots of lysates from HEK293T cells co-overexpressing Flag-tagged MPP9 (401–800 aa) and TTBK2-GFP-WT or TTBK2-GFP-KD after treatment with λ-PPase. Tubulin was used as a loading control. d Mass spectrometric analysis showing phosphorylation sites of MPP9 at S629 and S636. e Immunoblots of lysates from HEK293T cells co-overexpressing TTBK2-GFP and Flag-MPP9 (401–800 aa)-WT, or the indicated unphosphorylatable mutants. Tubulin was used as a loading control. f GST-MPP9 (401–800 aa)-WT, or the indicated mutants were subjected to a TTBK2 kinase assay in vitro followed by autoradiography. Coomassie blue (CBB) staining showed GST-tagged proteins. Relative amounts of phosphorylated MPP9 signals were quantified. g Lysates of HEK293T cells co-overexpressing Flag-tagged MPP9-WT or the S629A mutant and TTBK2-GFP were subjected to immunoprecipitation (IP) and immunoblotting with anti-Flag and anti-Phospho-S629 antibodies. Relative amounts of S629-phosphorylated MPP9 were quantified. h Immunostaining of S629-phosphorylated MPP9 (red) and Flag in Flag-MPP9 (green, upper) or Flag-CEP170 (green, lower) overexpressing hTERT RPE-1 cells. i Immunostaining of S629-phosphorylated MPP9 (red) and Flag in Flag-MPP9 (green) or Flag-MPP9–629A (green) overexpressing hTERT RPE-1 cells after depleting MPP9. j Quantifications of S629-phosphorylated MPP9 signals in i (n = 50 cells for each group). k Immunostaining of S629-phosphorylated MPP9 (green) and Centrin-3 (red) in control- or TTBK2-siRNA transfected hTERT RPE-1 cells. l Quantifications of S629-phosphorylated MPP9 signals in k (n = 50 cells for each group). m Lysates of control-siRNA or TTBK2-siRNA transfected hTERT RPE-1 cells after serum starvation were subjected to immunoprecipitation and immunoblotting with the indicated antibodies. Relative amounts of S629-phosphorylated MPP9 were quantified and normalized to MPP9. For j, l, bars represent the means ± S.E.M for three independent experiments. ***p < 0.001, as determined by unpaired two-tailed Student’s t-test. Scale bars: 1 μm (h, i, k)