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Fig. 1. A proteomic screen for ROCK-regulated changes during PMA-induced apoptosis. D2 cells were pre-treated with or without 20 µM Y27632 for 1 hour in serum-free medium on hydrogel-coated dishes prior to treatment with 32.4 nM PMA. (A) After PMA treatment for 8 hours, cell viability was determined by the Trypan Blue exclusion method. NT, cells without either PMA or Y27632 treatment. (B) Phase-contrast micrographs of D2 cells after 4 hours of PMA treatment as indicated. (C) Following treatment with PMA for 4 hours, the cytosolic proteins of cells treated with and without Y27632 were prepared and labeled with Cy3 and Cy5, respectively. Two sets of these labeled lysates each containing 100 µg of proteins were pooled together and separated on a single 2DE gel. Green indicates Cy3-tagged proteins, red Cy5-tagged proteins and yellow colocalization of the spots. (D) A higher power image of spots corresponding to hnRNP C1/C2. (E) Whole cell extracts of D2 cells treated as in C were separated by 2DE and the gels were silver stained. Red dashed line outlines the spots corresponding to hnRNP C1/C2.
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