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. 2018 Jan 23;8:1410. doi: 10.1038/s41598-018-19471-2

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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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Group-I PAKs induce direct phosphorylation of HACE1. (a) In vitro [γ ³²P]-ATP kinase assay using recombinant 6His-HACE1 and recombinant 6His-PAK1 analyzed by autoradiography and Coomassie Brilliant Blue (CBB) staining. (b) MCF12A and HUVEC protein lysates analyzed by immunoblot (IB) using anti-PAK1 and anti-PAK2 antibodies. IB: actin is used as a loading control. (c–e) Protein lysates from (c) HUVECs and (d,e) MCF12A cells transfected with siRNAs targeting (c,d) PAK2 or (e) PAK1 and plasmid expressing HA-HACE1 and myc-Rac1(Q61L) and analyzed by IB using the indicated antibodies. (f) Protein lysates from MCF12A cells transfected with HA-HACE1, either intoxicated with CNF1 for 16 hours or co-transfected with myc-Rac1(Q61L), and treated with FRAX597 at the indicated concentration for 16 hours before IB analysis. (g,h) Protein lysates from MCF12A cells transfected with HA-HACE1, myc-Rac1(Q61L) and GST-KID2 or Flag-PAK1K141A and analyzed by IB. IBs in (a–h) are cropped from the full-length blots shown in Supplemental Figure S11.