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Fig. 1. A subset of Golgi-associated transport factors are cleaved in apoptotic extracts. (A) Isolated rat liver Golgi membranes were incubated at 37°C with apoptotic HeLa cytosol in the absence or presence of 2 µM Ac-DEVD-CHO (D) for the times indicated. Membranes were pelleted and membrane and supernatant (cytosol) fractions analysed by immunoblotting with antibodies to giantin, GM130, syntaxin 5, GS28, or mannosidase I (Mann-1). Full-length proteins (FL) and caspase cleavage products (*) are marked. The major giantin cleavage products are denoted P1-P3. Minor giantin cleavage products are indicated with filled circles. (B) Golgi membranes were incubated at 37°C for 4 hours with control HeLa cytosol, apoptotic cytosol, or apoptotic cytosol in the presence of 2 µM Ac-DEVD-CHO. Samples were fractionated as in A and analysed by immunoblotting with antibodies raised against fragments encompassing the entire protein (mix) or the N terminus (NT) of giantin. The x indicates an unknown cytosolic protein that cross-reacts with the N-terminal antibody. (C) Golgi membranes were incubated at 37°C for 6 hours with control HeLa cytosol, apoptotic cytosol, or apoptotic cytosol in the presence of 2 µM Ac-DEVD-CHO. Samples were fractionated as in A and analysed by immunoblotting for a number of Golgi-associated proteins, as indicated. Blotting with three different polyclonal antibodies to p115 gave identical results.
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