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Fig. 3. Colocalization of SI mutants with ER, ERGIC and cis-Golgi in transfected COS-1 cells. COS-1 cells were transfected with pSI Ala1-6-YFP and incubated at 37°C (A-C) for 48 hours before fixation. In some experiments the cells were cultured at 20°C 1 day post-transfection and then overnight at 20°C followed by a temperature shift to 37°C for 4 hours (D). The subcellular localization of the YFP fusion proteins was monitored utilizing antibodies directed against calreticulin (A), ERGIC 53 (B) and GM130 (C). The cells were analysed by confocal microscopy on a Leica TCS-SP2 microscope. YFP fluorescence is indicated in green and a secondary Alexa Fluor 633-conjugated antibody was used for immunodetection of the compartment-specific antibodies (red). Arrows indicate YFP fluorescence on the plasma membrane. n, nucleus. Bars, 25 µm.
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