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. 2017 Aug 25;7:9524. doi: 10.1038/s41598-017-10210-7

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© The Author(s) 2017

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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The anti-pY87 signal at the focal adhesions corresponds to PKD2. (A) HeLa cells were infected with lentivirus expressing non-target shRNA (scr-shRNA), or two independent shRNA sequences targeting PKD2 (PKD2-shRNA#1, PKD2-shRNA#2). After 48 hours, cells (6000/channel) were reseeded in ibidi channel μ-slides. Cells were subjected to immunofluorescence analysis to determine the localization of endogenous pY87-PKD2 (secondary antibody used: Alexa Fluor 488) and F-actin (phalloidin). Scale bar indicates 10 μm. (B) Shows quantitation for the number of pY87-PKD2-positive adhesions per cell with scr-shRNA (n = 73 cells), PKD2-shRNA#1 (n = 63 cells) and PKD2-shRNA#2 (n = 64 cells). Error bars represent standard error of the mean (SEM). *Indicates statistical significance (p < 0.05) as compared to cells expressing scr-shRNA. (C) Cell lysates from A were evaluated by Western blotting for the expression of PKD2 and β-actin. All experiments have been performed at least three times with similar results. Uncropped blots are shown in Supplemental Figure S7. (D) HeLa cells were infected and reseeded in ibidi channel μ-slides, as described in (A) Cells were subjected to immunofluorescence analysis to determine the localization of endogenous pY87-PKD2 and paxillin. Scale bar indicates 10 μm. (E) Shows quantitation for the number of paxillin-positive adhesions per cell with scr-shRNA (n = 40 cells), PKD2-shRNA#1 (n = 40 cells) and PKD2-shRNA#2 (n = 40 cells). Error bars represent standard error of the mean (SEM). *Indicates statistical significance (p < 0.05) as compared to cells expressing scr-shRNA.