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. 2008 Jun 30;181(7):1179–1193. doi: 10.1083/jcb.200709086

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© 2008 Kumari et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 3. — The AChR–αBTX complex is trafficked to late endosomes in CHO and C2C12 cells. (A) CHO-K1/A5 cells were labeled with Cy3αBTX for 1 h at 4°C and were chased for 0, 2, 4, or 6 h at 37°C, fixed, and stained with the indicated antibodies to organelle-specific markers EEA1 (early endosomes) and LAMP1 (lysosomes). Images of Cy3αBTX (red) and organelle-specific markers (green) were collected from single slices on a confocal microscope and color combined. Note that Cy3αBTX bound to AChR is initially extensively colocalized with EEA1, and, after a chase of 6 h, it is located with the late endosomal marker. Insets show magnified images of the boxed areas for αBTX (red outline) with EEA1 or LAMP1 (green outline) and a merge of the two (white outline). (B) The histogram represents the colocalization index of internalized αBTX with EEA1 or LAMP1 at the indicated time points depicted as a mean of the percentage of internalized αBTX colocalizing with the indicated organelle markers per cell (see Materials and methods). Error bars represent SEM. (C) C2C12 cells were labeled with Cy3αBTX as in A, chased for 0, 2, or 6 h, washed, fixed, and processed for indirect immunofluorescence for EEA1 (top) or lysobisphosphatidic acid (bottom). Insets are magnified images of the boxed areas for αBTX (red outline) and EEA1 or LBPA (green outline). (D) CHO-K1/A5 cells transfected with GFP-GPI (left) or caveolin 1–GFP were labeled with Cy3αBTX for 1 h on ice, chased at 37°C for 2 h, and subsequently imaged live. Images represent a single confocal section of live cells showing colocalization between αBTX (red) and GFP-GPI or caveolin 1 (green). Note that the tubular structures of αBTX (insets) are devoid of GFP-GPI or caveolin 1. Bars, 10 μm.