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. 2003 Mar;23(5):1764–1774. doi: 10.1128/MCB.23.5.1764-1774.2003

FIG. 2.

FIG. 2.

Mitogen-dependent regulation of cyclin D1-Hsc70 binding. (A) Wild-type NIH 3T3 cells were used as an asynchronous culture (asyn) or synchronized by serum deprivation for 36 h and stimulated to reenter the cell cycle by addition of 10% fetal calf serum. Cell lysates were prepared at the indicated intervals (bottom) and immunoprecipitated (IP) with normal rabbit serum (NRS) or a cyclin D1-specific monoclonal antibody (D1). Precipitated proteins were then subjected to immunoblot analysis with antibodies specific for Hsc70 (top) or cyclin D1 (bottom). (B) D1-3T3 cells were synchronized as in panel A. Cell lysates were prepared at the indicated intervals (bottom) and precipitated with normal rabbit serum (NRS) or the M2 monoclonal antibody and subjected to immunoblot analysis with antibodies specific for Hsc70 (top panel) or cyclin D1 (bottom panel). (C) Cell lysates prepared as for panel A were subjected to direct Western analysis with an Hsc70-specific antibody. (D) Whole-cell lysates prepared from asynchronous D1-3T3 cells or D1-3T3 cells cultured in medium containing 0.1% fetal calf serum for the indicated intervals were precipitated with normal rabbit antiserum (NRS) or with the M2 monoclonal antibody. Precipitates were immunoblotted with either Hsc70 (top panel) or cyclin D1 (lower panel) antibodies. The arrow indicates the point at which cells were placed in medium containing 0.1% serum.