Figure 1.
The association between P/CAF and cyclin D1 in vivo. (A) COS cells were transfected with the indicated combinations of pCX-FLAG-P/CAF (F-P/CAF) and pRcCMV-cyclin D1-HA, pCMX-Gal4-cyclin D1, and pCMX-Gal4. Immunoprecipitations were carried out with antibodies against the FLAG epitope or the Gal4 DNA-binding domain. Precipitated proteins were separated by SDS/PAGE on a 10% gel, transferred to a polyvinylidene difluoride membrane, and analyzed by Western blotting with antibodies against cyclin D1 or the FLAG epitope. (Lower) Whole-cell lysate equivalent to one-tenth of the input for each immunoprecipitation. (B) SAOS-2 cells were transfected with the indicated combinations of FLAG-P/CAF, FLAG-ER, and cyclin D1 expression plasmids. They then were metabolically labeled with [35S]methionine for 4 hr, and immunoprecipitations were carried out with the M2 antibody. Precipitated proteins were resolved on a 10% gel and detected by autoradiography. (C) COS cells were transfected with pRcCMV-cyclin D1-HA, pRcCMV-cyclin D2-HA, or pRcCMV-cyclin D3-HA, plus either pCX-FLAG-P/CAF or the empty vector. Immunoprecipitations were carried out with anti-FLAG antibodies. Proteins were detected by Western blotting with an antibody against the HA-epitope.