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Fig. 5. Internalized ErbB2 colocalizes with Alexa-Fluor-594-CtxB, GPI-anchored proteins and dextran in GA-treated SK-BR-3 cells. Cells were pretreated with GA for 1 hour, subjected to antibody and/or toxin binding, warmed for 5 minutes, acid-stripped and fixed. (A) Fl-anti-ErbB2 (green) and Alexa-Fluor-594-CtxB (0.5 µg/ml; red) were bound to cells. A merged maximum-intensity projection image of a deconvolved z-stack is shown. Asterisks delimit the region shown enlarged in B (ErbB2, left; Alexa-Fluor-594-CtxB, middle; merged image, right). (C) Fl-anti-PLAP Fab fragments and Rh-anti-ErbB2 antibodies were bound to cells. A deconvolved image from a z-stack, showing part of the edge of one cell, is shown. ErbB2, left; PLAP, middle; merged image, right. (D) Fl-anti-ErbB2 was bound to cells, which were warmed with 1 mg/ml FluoroRuby dextran. An epifluorescence image, showing part of the edge of one cell, is shown. ErbB2, left; dextran, middle; merged image, right. Scale bars: 10 µm (A); 5 µm (D; applies to B-D). (E) Colocalization of ErbB2 and CtxB, or ErbB2 and PLAP, in cells treated as in A-C (except that internalization was for 2 minutes) was quantified. To measure the colocalization of ErbB2 and Thy1.1, SK-BR-3 cells transfected with Thy1.1 were treated with GA for 1 hour. Fl-anti-ErbB2 and Alexa-Fluor-594-anti-Thy1 Fab fragments were bound on ice, and cells warmed for 2 minutes. Residual surface-bound antibodies were acid-stripped before fixation, visualization and quantification.
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