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. 2021 Oct 15;12(10):951. doi: 10.1038/s41419-021-04241-2

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© The Author(s) 2021

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. 4 — A Left: Typical images of immunofluorescence evaluating p27 expression (green) in parental (top) and resistant (bottom) SW480 cells. Staining of F-Actin (red) and nuclei (To-Pro-3, blue) was used to evaluate p27 subcellular localization. Right: Graph reports the quantification of p27 expression (expressed as arbitrary units) in nuclear and cytoplasmic compartments. Each dot corresponds to one cell. Mann–Whitney test was used for statistical analysis. B Western blot analysis evaluating the expression of the indicated cell-cycle proteins in SW480 cells treated or not with Palbo (2 μM, 24 h) and resistant cells cultured in presence of 2 μM Palbo. C Expression of the indicated proteins in the nuclear (N) and cytoplasmic (C) fractions of SW480 cells treated or not with Palbo (2 μM, 24 h) and resistant cells, as indicated. D Kinase assay evaluating the activity of endogenous CDK2 immunoprecipitation from parental SW480 treated or not with 2 µM Palbo for 24 h and Palbo-resistant cells cultured in the presence of 2 µM Palbo. In the kinase assay recombinant Histone H1 (^32P-HH1) was used as substrate. Input indicates the expression of CDK2 in corresponding cell lysates and vinculin was used as loading control. Immunoglobulin G (IgG) represents the control IP using an unrelated antibody. E Co-immunoprecipitation (IP) analysis of endogenous thyrosin phosphorylated proteins (pY) in SW480 parental cells, treated or not with Palbo (2 μM, 24 h), and Palbo-resistant cells, serum starved (0) and released with FBS for 15’, as indicated. IP indicates the expression of pY-p27. Input stands for the expression of indicated proteins in corresponding cell lysates and vinculin was used as loading control. F Immunoprecipitation (IP) analysis of endogenous phosphorylated p27^Y88 in SW480 parental and resistant cells, serum starved (0) and released with FBS for 15’. Immunoglobulin G (IgG) represents the control IP using an unrelated antibody. Input indicates the expression of total p27 in corresponding cell lysates and vinculin was used as loading control. G Expression of the indicated proteins and phospho-proteins in cell lysates used in the IP assays shown in E and F. H Graph reports the cell viability of parental (black line) and resistant (pink line) SW480 cells, treated for 72 h with increasing doses of Cetuximab and evaluated using the MTS assay. Values from untreated cells represent 100% viability. Data represent the mean (±SD) of three independent experiments performed in sextuplicate.