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Fig. 2. PKC isoform expression and subcellular redistribution. (A) CHO{alpha}Vß3 cells were serum-starved, detached and kept in suspension for 1 hour, then replated in LM609-coated wells for the indicated times. Aliquots of cells were left untreated (suspension) for baseline detection, or treated with 10-7 TPA for 5 minutes to activate classical and novel PKCs. Cell proteins were fractionated as described in Materials and Methods, resolved by SDS-PAGE and western blotted with the indicated anti-PKC (diluted 1:800), or with anti-actin (diluted 1:1000) antibodies for normalization. (B) Osteoclasts were lifted, replated in LM609-coated wells for 30 minutes and processed for PKC or actin detection as described in A. C, cytosolic; M, membrane; I, Triton-X-100-insoluble fractions; Susp, cells in suspension; LM609, adhesion to LM609 for 30 minutes. Similar results were obtained in three independent experiments.





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