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. 2014 May 1;127(9):2071–2082. doi: 10.1242/jcs.144923

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© 2014. Published by The Company of Biologists Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Fig. 1. — Rab20 association with early phagosomes in IFN-γ-activated macrophages. (A) Analysis of the association of endogenous Rab20 with phagosomes in RAW264.7 macrophages. RAW264.7 macrophages were incubated with 3-µm IgG-coated beads for 1 h and 15 min of chasing, and were subsequently fixed and stained for Rab20. Nuclei were stained with Hoechst 33258 and are shown in blue. Magnified images show the association of Rab20 with phagosomes. Scale bar: 10 µm. (B) Quantitative analysis of the association of endogenous Rab20 with phagosomes at the indicated time-points. Data show the mean±s.e.m. of one representative experiment out of three independent experiments. At least 100 phagosomes were analyzed in each experiment. ***P≤0.001 (Student's two-tailed unpaired t-test). (C) Analysis of the association of endogenous Rab20 with phagosomes in bone marrow macrophages (BMM) as in A. Scale bar: 10 µm. (D) Quantitative analysis of the association of endogenous Rab20 with phagosomes in BMM as in B. (E) Western blot analysis of the association of endogenous Rab20 with phagosomes in RAW264.7 macrophages that were untreated or treated with 200 U/ml IFN-γ for 16 h. Latex-bead phagosomes were isolated from untreated or IFN-γ-treated RAW264.7 macrophages after 15, 30 and 60 min of chase. Rab20 and Rab5a levels were detected and actin was used as the loading control. (F) Quantification of the association of Rab20 with phagosomes from E, calculated relative to actin and normalized to the value of untreated macrophages at 15 min. Data show the mean±s.e.m. of three independent experiments. *P≤0.05 (Mann-Whitney test). (G) IFN-γ-treated RAW264.7 macrophages were incubated with 1-µm IgG-coated beads for 1 h and then processed for cryosectioning. Rab20 was detected by using a rabbit anti-Rab20 antibody followed by labeling with 10-nm Protein-A–gold. Arrowheads indicate the presence of Rab20 at the phagosomal membrane. (H) Quantitative analysis of the number of phagosomes positive for Rab20 in cells treated with IFN-γ or left untreated.