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Fig. 5. Colocalization of the 60S ribosomal subunit with translocon protein Sec 61. (A-C) Sec 61, recognized by polyclonal Sec 61 antibodies, was detected using FITC-labeled secondary antibodies. 60S ribosomal subunit, recognized by polyclonal antibodies, was detected using Texas-Red-labeled secondary antibodies. No staining was seen in the absence of primary antibodies (data not shown). Cells were analyzed using a Zeiss LSM 510 confocal laser scanning system connected to a Zeiss Axiovert 200 M. Images were collected separately for each channel (Texas-Red at 563 nm and FITC at 488 nm excitation) and merged as indicated. The pictures were taken after a 1-hour treatment under control conditions (A), with 200 µM puromycin (B) or with 1.8 mM cycloheximide (C). Bar, 20 µm. (D) Correlation coefficients calculated according to reported methods (Manders et al., 1993). Puromycin slightly reduced colocalization of the Sec61 and ribosome labels. (E-G) Transmitted electron micrographs of LNCaP cells in control conditions (E), or after a 1-hour treatment with 200 µM puromycin (F) or 1.8 mM cycloheximide (G). Bar, 200 nm.
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