Article Title: Cytoplasmic Localization of Human cdc25C during Interphase Requires an Intact 14-3-3 Binding Site
Figure Lengend Snippet: cdc25C is present in the cytoplasm of asynchronously growing human cells. (A) Two-milligram aliquots of EBC extracts prepared from U-2OS cells were immunoprecipitated with cdc25C-specific antibodies TC14 (lanes 2 and 7), TC15 (lanes 3 and 8), TC113 (lanes 4 and 9), and TC19 (lanes 5 and 10) or the Myc antibody 9E-10 (lanes 1 and 6). The immune complexes resolved in SDS–7.5% polyacrylamide gels followed by Western blotting with either a mixture of TC14, TC15, and TC19 (lanes 1 to 5) or an anti-cdc25C rabbit polyclonal antibody (Santa Cruz) (lanes 6 to 10). The position of cdc25C, which migrated as a doublet, is indicated by the bracket. The arrow indicates the immunoglobulin heavy chain. The positions of the molecular weight markers are indicated in kilodaltons to the left of each gel. B. U-2OS (lanes 1, 4, and 7), MRC-5 (lanes 2, 5, and 8), and WI-38 (lanes 3, 6, and 9) cells were harvested by trypsinization, and the cell pellets were boiled in 2% SDS; 50 (lanes 1 to 6) or 25 (lanes 7 to 9) μg of protein extract was separated in an SDS–10% polyacrylamide gel, and then Western blotting was performed with the indicated antibodies. The immune complexes were detected by using the Pierce Supersignal system. TC14, TC15, and TC113 all recognized a specific doublet at ∼55 kDa in each of the different cell types. In addition to this band, they specifically recognized a band at ∼47 kDa (open arrow) in U-2OS cells. On longer exposures, this band was also detected in MRC-5 and WI-38 cells. No other specific signal was detected with these antibodies. (C) Two-milligram aliquots of whole-cell (lanes 1 and 4), cytoplasmic (lanes 2 and 5), or nuclear (lanes 3 and 6) extracts prepared from U-2OS cells were immunoprecipitated with either an anti-Rb antibody (MAb 245; Pharmingen) (lanes 1 to 3) or an anti-cdc25C antibody (TC113) (lanes 4 to 6). The immune complexes were resolved in an SDS–7.5% polyacrylamide gel and Western blotted with MAb 245 (lanes 1 to 3) or a mixture of TC14, TC15, and TC19 (lanes 4 to 6). The cdc25C doublet is indicated by the bracket. The thick arrow indicates the immunoglobulin heavy chains; the position of Rb on the gel is indicated by the thin arrow. (D) Two-milligram aliquots of whole-cell (lane 1), cytoplasmic (lanes 2 and 3), or nuclear (lane 4) extracts prepared from U-2OS cells were immunoprecipitated with the cdc25C MAb (TC113). The cytoplasmic extracts in lane 3 were prepared in the presence of a cocktail of phosphatase inhibitors (10 μM cypermethrin, 200 μM dephostatin, 200 nM okadaic acid, and 25 nM tautomycin). cdc25C is indicated by a bracket; the arrow indicates the position of the immunoglobulin heavy chain.
Article Snippet: All four cdc25C antibodies specifically immunoprecipitated a doublet that migrated at approximately 55 kDa in U-2OS cells upon Western blotting with either a mixture of the cdc25C MAbs or an anti-cdc25C rabbit polyclonal antiserum (C-20; Santa Cruz) raised against the C terminus of cdc25C (Fig. A, lanes 2 to 5 and lanes 7 to 10, respectively).
Techniques: Immunoprecipitation, Western Blot, Molecular Weight