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. 2019 Apr 23;9:6394. doi: 10.1038/s41598-019-42767-w

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© The Author(s) 2019

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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Pin1 interacts with TAZ in vitro and in vivo. (A) Pin1 interacts with TAZ in vitro. 200 µg of cell lysates from FLAG-tagged-TAZ plasmid transfected HEK293 cells were precleared with 50% GSB beads overnight at 4 °C. After then, supernatants were mixed with 5 µg of GST or Pin1-GST and incubated for 2 hrs followed by addition of 20 μl of 50% GSB beads for another 1 hr. The beads were then washed, eluted by 2XSDS sample buffer and subjected to western blotting against anti-FLAG antibody. Ponceau-S staining shows the equal amount of fusion protein used for pull down. 1/10 input (20 µg) represents 1/10 of protein lysate used for pull down. (B) Pin1 interacts with TAZ in vivo. HEK293 cells were transfected with HA-tagged-Pin1-WT or FLAG-tagged-TAZ plasmids alone or together. The cells were harvested in 1%NP-40 lysis buffer. After checking the expression level of HA-tagged-Pin1-WT or FLAG-tagged-TAZ, equal amount of cell lysates were subjected to co-immunoprecipitation assays using anti-HA antibody and immublotting analysis were performed using anti-FLAG or anti-HA antibody respectively. (C) WW domain of Pin1 interacts with TAZ in vitro. GST pull down assay was carried out as described above using the lysate from FLAG-tagged-TAZ transfected HEK293 cells and GST or Pin1-WT-GST, Pin1-WW-GST or Pin1-PPIase-GST separately (D) Pin1-WW domain is necessary for interaction of Pin1with TAZ in vivo. Co-IP was carried out as described above by using the cell lysate from FLAG-tagged-TAZ or HA-Pin1-WT,- WW or -Pin1-PPIsae alone or together transfected HEK293 cells. Anti-HA antibody was used for immunoprecipitation and immublotting analysis were performed using anti-FLAG or anti-HA antibody respectively. (E) Pin1-W34A mutant abolish its interaction with TAZ in vitro, GST pull down assay was carried out as mentioned in Fig. 1A using the FLAG-TAZ transfected HEK293 cell lysates and GST, Pin1-WT-GST or Pin1-W34A-GST. (F) Mutation at W34A at WW domain of Pin1 abolish the interaction of Pin1 with TAZ in vivo. HEK293 cells were transfected with HA-tagged-Pin1-WT, -W34A or FLAG-tagged-TAZ plasmids alone or together. The cells were harvested in 1%NP-40 lysis buffer and co-immunoprecipitation was carried out as described above.