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. 2018 Sep 20;9:3825. doi: 10.1038/s41467-018-06367-y

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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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Fig. 5 — Clathrin-coated plaques locally regulate receptor-dependent Erk signaling. a HeLa cells seeded on collagen-coated glass were starved for 4 h and then treated or not with 10 ng/ml EGF for 5 min alone or added after 30 min preincubation with 10 µM Gefitinib. Cells were then fixed and stained for α-adaptin and phosphotyrosines. The arrowhead points to one CCP and the star marks a plaque. Scale bar: 2 μm. b Quantification of phosphotyrosines accumulation at plaques or CCPs in the indicated conditions. Results are expressed as mean ± SD (*P < 0.05, **P < 0.001, one-way analysis of variance—ANOVA. n = 3. Number of structures analyzed per condition: Starved 160 plaques; EGF: 452 plaques, EGF + Gefitinib: 301 plaques, CCPs/EGF: 200 pits). c Western-blot analysis of phospho-Erk (P-Erk) levels in starved HeLa cells treated with control or β5-specific siRNAs as indicated, and stimulated for the indicated time with 10 ng/ml EGF. Total-Erk was used as a loading control (representative image of three independent experiments). d Densitometry analysis of bands obtained in western-blots as in c. Results are expressed as mean ± SD (*P < 0.05, ANOVA. n = 3). e HeLa cells treated with β5-specific siRNAs were transfected with plasmids encoding for TfR-mCherry and EGFR-GFP and seeded on anti-mCherry antibodies-coated glass. Cells were serum-starved for 4 h and then treated with 10 ng/ml EGF for 470 s. Scale bar: 0.5 μm. Arrowheads point to plaques positive for EGFR-GFP. f HeLa cells treated with β5-specific siRNA were transfected with plasmids encoding for TfR-mCherry and HGFR-GFP and seeded on anti-mCherry antibodies-coated glass. Cells were serum-starved for 4 h and then treated with 50 ng/ml HGF for 570 s. Scale bar: 0.5 μm. Arrowheads point to plaques positive for HGFR-GFP. g HeLa cells treated with β5-specific siRNA were transfected with plasmids encoding for TfR-mCherry and seeded on anti-mCherry antibodies-coated glass. Cells were serum-starved for 4 h and then treated with 10 ng/ml EGF for 5 min prior to fixation and staining for phosphotyrosines. Scale bar: 1.5 μm. Arrowheads point to plaques positive for P-Tyr