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. 2015 Nov 6;10(11):e0142062. doi: 10.1371/journal.pone.0142062

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© 2015 Hirano, Kanno

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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Fig 4 — (A) GFP-MARCO-CHO cells were incubated with or without (NH₄)₂CO₃ and the cells were loaded with pHrode Red AM. The cells were washed and cultured in HEPES-buffered HBSS for 2h. The acidity of lysosomes was neutralized by (NH₄)₂CO₃. (B) Macropinocytic inclusions (arrows) did not co-localize with lysosomes. The control GFP-MARCO-CHO cells were fixed and stained with anti-LAMP2 antibody (2^nd Ab: Alexa Fluor® 594-labeled goat anti-mouse IgG). (C) Co-localization of GFP-MARCO and LAMP2 in autophagic puncta. The cells were grown for 15 hr in the presence of 50 μM chloroquine. See also the legend to Fig 4 (B) for immunofluorescent staining. (D) GFP-MARCO-CHO cells were cultured for 15 hr in fresh F12 medium in the presence or absence (Control) of 4 mM (NH₄)₂CO₃. The supernatant of the cellular lysate was analyzed by SDS-PAGE followed by western blotting for the detection of GFP. The membrane was reprobed with POD-tagged anti-tubulin to serve loading control.