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. 2018 Mar 26;9:1234. doi: 10.1038/s41467-018-03628-8

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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 — STAT1 is a novel Intu-interacting protein at the basal body/centriole area. a To identify Intu-interacting proteins, Intu was transiently expressed in BUMPT cells, followed by Intu pulldown by the TAP assay. Eluted proteins from the TAP were subjected to SDS-PAGE and silver staining. One band at ~90 kDa was analyzed by mass spectrometry identifying two peptides of STAT1 (in arrowed box). b Intu constructs (or empty vector) were transiently transfected into BUMPT cells and STV-pulldown assay was performed to determine the co-precipitation of Intu and STAT1. c Intu-mycDDK was transfected into 293FT cells to collect lysate for immunoprecipitation of STAT1 with anti-STAT1 antibody or non-immune IgG (control). The precipitates were then analyzed for STAT1 and myc-tagged Intu to show their co-precipitation or interaction. d Co-immunoprecipitation of Intu and STAT1 in kidney tissues of PT-Intu-KO or WT mice after renal IRI. e Intu deletion mutants were transfected into BUMPT cells. After STV-pulldown, the Intu N-terminus containing PDZ domain (CBP-SBP-Intu ∆268-942) precipitated endogenous STAT1 while the mutants lacking this domain did not. f Intu and STAT1 interaction during IFN-γ, azide, and cisplatin treatment. 293FT cells were transfected with Intu constructs (CBP-SBP-Intu) and subjected to different treatments. Cell lysate was collected for STV-pulldown assay to show that more STAT1 was pulled down by Intu in treated 293FT cells. g Double immunofluorescence of Intu and γ-tubulin (marker of basal body/centriole) showing their co-localization in cultured mouse (BUMPT) and rat (RPTC) kidney proximal epithelial cells at different cell cycle phases. h Double immunofluorescence of Intu and Ac-tubulin (marker of cilia) showing the localization of Intu at the base of primary cilia. i Double immunofluorescence of Phospho-STAT1^S727 and γ-tubulin revealed their co-localization, while double immunofluorescence of total STAT1 and γ-tubulin showed the distribution of total STAT1 around basal body/centrioles. In three panels g–i, cells were also stained with DAPI to show nuclear morphology in merged images. Arrow-head: basal body/centriole. Scale bar: 10 µm