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. 2020 Jun 4;11:2817. doi: 10.1038/s41467-020-16309-2

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© The Author(s) 2020

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. 7 — a Immunoprecipitation (IP) of endogenous AGO2 upon EGF stimulation (5′) in the indicated cancer cells expressing wild-type RAS followed by immunoblot analysis of KRAS. For MCF7 cells, endogenous co-IP analysis was performed using both AGO2 and KRAS-specific antibodies. For each cell line and panel in this figure, MAPK activation and levels of various proteins are shown as input blots. b IP of endogenous AGO2 upon EGF stimulation (5′), in the indicated cancer cells harboring different KRAS mutations, followed by immunoblot analysis of KRAS. c Co-IP and immunoblot analysis of RAS and AGO2 upon EGF stimulation of HEK293 (wild-type KRAS) cells expressing FLAG-AGO2 or (d) DLD-1 (KRAS^G13D) cells in the presence or absence of erlotinib. e EGF stimulation and RAS co-IP analysis in HEK293 (wild-type KRAS) and f MIA PaCa-2 (KRAS^G12C) cells expressing FLAG-tagged AGO2 (wild-type or Y393F). IP of endogenous AGO2 upon H₂O₂ treatment (4′), in H358 (g) and MIA PaCa-2 (h) cells harboring KRAS mutations, followed by immunoblot analysis of KRAS. i H₂O₂ treatment and KRAS-AGO2 co-IP analysis in MIA PaCa-2 (KRAS^G12C) cells expressing FLAG-tagged AGO2 (wild-type or Y393F). Numbers on the left of the immunoblots in this panel indicate protein molecular weights in kDa. j Left panels, Representative images of single target (RAS or AGO2) and RAS-AGO2 interaction PLA in wild-type RAS expressing PC3 cells across the indicated cell culture conditions. Right panels, Representative images of PLA to detect RAS-AGO2 interaction in wild-type RAS expressing MCF-7 (panel I) and oncogenic KRAS expressing HCT116 (panel II) and H358 (panel III) cells grown in the indicated culture conditions. PLA signals appear as red dots around DAPI stained nuclei in blue. Scale bar, 50 µm.