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Figure 5


Fig. 5. Immunoprecipitation of {alpha}-tubulin from apoptotic lysates. (A) Jurkat cells pre-labelled with [35S]methionine were incubated with unlabelled effector cells at an E:T of 1:1. Specific full-length {alpha}-tubulin is indicated by * in lane 1, and cleaved product by ** in lane 2. Non-specific products are shown in lanes 3 and 4. (B) Jurkat cell lysates were separated into fractions containing soluble {alpha}-tubulin (S) and fractions containing polymerized microtubules (P) (see Materials and Methods for details). GrB was added to 5 µM (lanes 2 and 5) and 10 µM (lanes 3 and 6). Protein products were separated by 10% SDS-PAGE and analyzed by western blot with anti {alpha}-tubulin. (C) Jurkat and CTL (J + CTL) were incubated at an E:T of 1:1 prior to solubilization. The total protein solution (T) was separated into fractions containing soluble {alpha}-tubulin (S) and fractions containing polymerized microtubules (P) (lanes 10-12). The efficiency of fractionation to the soluble phase was verified by incubation of Jurkat cells with the microtubule depolymerizing agent nocodazole (Noc, lanes 4-6). The efficiency of fractionation to the polymerized phase was verified by incubation of Jurkat cells with the microtubule polymerizing agent paclitaxel (Pac, lanes 7-9). Protein products were separated on a 12% SDS-PAGE and analyzed by western blot with anti {alpha}-tubulin.





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