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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. 4 — MPP9 recruits CEP97 and CP110 at the distal end of the mother centriole in hTERT RPE-1 cells. a Immunoblots showing depletion of MPP9, CP110, or CEP97 by siRNA in hTERT RPE-1 cells. GAPDH was used as a loading control. b Immunostaining of acetylated-tubulin (Acet-Tub, red) and CP110 (green) in the control- or MPP9-siRNA-treated hTERT RPE-1 cells. c Quantification of the percentage of cells with the indicated number of CP110 dots from b. d Immunostaining of Acet-Tub (red) and CEP97 (green) in the control- or MPP9-siRNA-treated hTERT RPE-1 cells. e Quantification of the percentage of cells with the indicated number of CEP97 dots from d. f Immunoblots showing expression of the indicated proteins in MPP9-depleted hTERT RPE-1 cells after treatment with DMSO or MG132 for 4 h. Tubulin was used as a loading control. Relative amounts of MPP9, CEP97, and CP110 were quantified and normalized to tubulin. g Immunostaining of Centrin-3 (red) and CEP97 (green, upper) or CP110 (green, lower) after treating with DMSO or MG132 for 4 h in MPP9-depleted hTERT RPE-1 cells. h Immunostaining of CEP97 (red) and Flag-siRNA-resistant MPP9 (Flag-ResMPP9, green) or Flag-ResMPP9-△451-500 (green) in MPP9-depleted hTERT RPE-1 cells. i Immunoblots showing expression of the indicated proteins in hTERT RPE-1 cells transfected with MPP9-siRNA and Flag-siRNA-resistant MPP9 wild-type (Flag-ResMPP9-WT) or lacking 451–500 aa mutant (Flag-ResMPP9-△451–500). Tubulin was used as a loading control. Relative amounts of CEP97 and CP110 were quantified and normalized to tubulin. j Immunostaining of Centrin-3 (red) and Arl13B (green) after transfection of Flag-ResMPP9-WT or Flag-ResMPP9-△451–500 in MPP9-depleted hTERT RPE-1 cells. DNA was stained with DAPI (blue). k Quantification of ciliogenesis in MPP9-depleted hTERT RPE-1 cells overexpressing Flag-ResMPP9-WT or Flag-ResMPP9-△451–500. For c, e, k, bars represent the means ± S.E.M for three independent experiments. n.s., not significant, *p < 0.05, **p < 0.01, as determined by unpaired two-tailed Student’s t-test (c, e), and one-way ANOVA analysis (k). Scale bars: 5 μm (j main); 1 μm (b, d, g, h, j insets)