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Fig. 6. Characterization of the anti-PRL recycling pathway. Cells were stained for internalized anti-PRL at the 0, 10, 20 and 80 min time points as described for Fig. 1. All images are from the t=20 min time point. (A,B) Following fixation and permeabilization, cells were also stained with anti-TGN38 or anti-syntaxin 6. (C) Cell fixed at the t=20 min time point, without permeablization, showing internalized Alexa 488 labeled anti-PRL (green) and DiI-LDL (red) that had been chased into the lysosomes. (D). Cell fixed at the t=20 minute time point that had been internalized Alexa 488 anti-PRL (green) and Alexa 546 transferrin (red). Bar, 10 µm. DAPI (blue) was included in the mounting media. (E) The extent of colocalization between internalized anti-PRL and the respective markers was determined from complete z-series of cells and is expressed as the percent of pixels that were positive for anti-PRL and positive for marker out of the number of pixels that were positive for anti-PRL. Data given are means (±s.e.m.) from 12 to 20 cells per condition and 30-50 z-sections per cell.
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