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. 2020 Nov 30;11(11):1023. doi: 10.1038/s41419-020-03220-3

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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. 2 — A HEK293T cells were transiently cotransfected with C-terminally Flag-tagged SH2D4A and HA-tagged PHB1 or PHB2. Proteins were immunoprecipitated with anti-Flag antibodies and immunoblotted with anti-SH2D4A and anti-HA antibodies. One representative experiment out of three with similar outcome is shown. HuH7 cells were seeded on glass coverslips, fixated with PFA and immunofluorescence staining (B) and proximity ligation assay (PLA) of endogenous SH2D4A and PHB1 co-stained with Mitotracker (C) were performed. Confocal microscopic images were analyzed using Fiji software. Fluorescence intensity profiles are shown in the right panels and represent the fluorescence distribution along the small white lines in the respective merge images. Scale bars: 20 µm. D Immunoblot of SH2D4A, STAT3, and pSTAT3-Ser727 in nuclear (Nucl) and cytoplasmic (Cyto N) fractions obtained with NE-PER Nuclear and Cytoplasmic Extraction Kit, and mitochondrial (Mito) and cytoplasmic (Cyto M) fractions obtained with Mitochondria Isolation Kit for Cultured Cells (both Thermo Fisher Scientific). PARP was used as nuclear, β-Tubulin as cytoplasmic, and PHB1 as mitochondrial marker. *unspecific band.