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. 2019 Oct 28;8(11):63. doi: 10.1038/s41389-019-0172-9

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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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Fig. 6 — a PR55α was immunoprecipitated from 1 mg protein lysates of CD18/HPAF cells expressing Control-shRNA or PR55α-shRNA with anti-PR55α (100C1) rabbit IgG and probed by immunoblotting for the presence of PR55α, PP2A-C, and PP2A-A subunits with anti-PR55α (2G9), anti-PP2A-C (ID6), and anti-PP2A-A (H300) antibody, respectively. YAP and GAPDH in the lysate were measured by immunoblotting for YAP protein loading control and internal control for protein quantification, respectively. b PR55α was immunoprecipitated from the indicated protein lysates (1 mg per sample) with anti-PR55 (100C1) antibody. The obtained immunoprecipitates were divided into two halves: one half remained untreated (−) and the other half was treated with Shrimp Alkaline Phosphatase (SAP) at 10 units/ml (+) at 37 °C for 1 h. The resulting precipitates were rinsed once with cell lysis buffer and subjected to immunoblotting analysis for PR55α, YAP, and YAP-S127 with the antibody for PR55α (2G9), YAP (D24E4), and YAP-Ser127 phosphorylation (D9W2I), respectively. c PR55α were immunoprecipitated (IP) with anti-PR55α (2G9) antibody from CD18/HPAF cells stably transduced with empty vector, Flag-YAP, or Flag-YAP (5SA) mutant and immunoblotted (IB) using anti-PR55α (100C1) and anti-Flag (M2) antibodies. Lysates from the indicated cells were probed for Flag-YAP (input) and GAPDH (input) with specific antibodies by Western blotting. Intracellular distribution and co-localization of PR55α and YAP in pancreatic malignant and normal cells. The indicated cells were induced for the expression of PR55α-shRNA (CD18/HPAF) (d) or ectopic PR55α (HPNE) (e) by 2 µg/ml Dox for 3 days, and stained with anti-PR55α (100C1) and anti-YAP (1A12) antibodies, as described in the “Materials and methods” section. Images were analyzed for the cellular distribution of PR55α and YAP using a Zeiss-810 confocal laser-scanning microscope. Co-localization of PR55α and YAP in CD18/HPAF cells with/without PR55α-knockdown and in HPNE cells with/without ectopic PR55α expression were examined and shown as merged images (PR55α/YAP and MERGE). Scale bars, 50 µm