That CtIP levels are co-dependent on Mre11 and CDK2 suggests a func¬tional relationship may exist among these factors. Hence, we conducted a series of coimmunoprecipitation experiments to explore the possi¬bility that MRN and CDK2 function together in a multiprotein com¬plex. First, endogenous Mre11 was immunoprecipitated from MEFs, and both CDK2 and cyclin A were observed to coimmunoprecipitate (Fig. 3a). To address the specificity of the coimmunoprecipitations and potential complexes, we blotted for cyclin D1, which is present in G1 and not known to associate with CDK2. Cyclin D1 was not detected in the coimmunoprecipitate fractions (Fig. 3a). Conversely, upon pre¬cipitation of hemagglutinin (HA)-tagged CDK2 transiently expressed in human cells (HeLa), we detected Mre11, Rad50 and NBS1 (Fig. 3b). In addition, when cyclin A was precipitated, Mre11 was present in the coimmunoprecipitation eluate (Fig. 3c). Finally, in cells deficient for MRN (Mre11−/−), CDK2 maintained association with cyclin A (Fig. 3c). Together, these findings indicate that MRN associates in vivo with CDK2 and its S-phase binding partner cyclin A.
Next, endogenous Mre11 was immunoprecipitated from CDK2−/− MEFs to determine whether other CDK family members associate with Mre11. Whereas NBS1 was detected in the coimmunoprecipitation fraction, no CDKs were identified by western blot analysis with an antibody to the highly conserved cyclin binding PSTAIR helix (Fig. 3d). In addition, cyclin A was not detected. Thus, our coimmunoprecipitations appear to be quite specific, and they support the notion that MRN does not associate globally with CDK–cyclins. We interpret the minimal impact on CtIP levels in
CDK2−/− cells (Fig. 2d) to indicate that an alternative kinase acts without Mre11 interaction. However, we cannot rule out the pos¬sibility that either weak interaction occurs below our detection limit or an uncharacterized kinase interacts that is not recognized by the PSTAIRE antibody interacts with Mre11.
That CtIP levels are co-dependent on Mre11 and CDK2 suggests a func¬tional relationship may exist among these factors. Hence, we conducted a series of coimmunoprecipitation experiments to explore the possi¬bility that MRN and CDK2 function together in a multiprotein com¬plex. First, endogenous Mre11 was immunoprecipitated from MEFs, and both CDK2 and cyclin A were observed to coimmunoprecipitate (Fig. 3a). To address the specificity of the coimmunoprecipitations and potential complexes, we blotted for cyclin D1, which is present in G1 and not known to associate with CDK2. Cyclin D1 was not detected in the coimmunoprecipitate fractions (Fig. 3a). Conversely, upon pre¬cipitation of hemagglutinin (HA)-tagged CDK2 transiently expressed in human cells (HeLa), we detected Mre11, Rad50 and NBS1 (Fig. 3b). In addition, when cyclin A was precipitated, Mre11 was present in the coimmunoprecipitation eluate (Fig. 3c). Finally, in cells deficient for MRN (Mre11−/−), CDK2 maintained association with cyclin A (Fig. 3c). Together, these findings indicate that MRN associates in vivo with CDK2 and its S-phase binding partner cyclin A.Next, endogenous Mre11 was immunoprecipitated from CDK2−/− MEFs to determine whether other CDK family members associate with Mre11. Whereas NBS1 was detected in the coimmunoprecipitation fraction, no CDKs were identified by western blot analysis with an antibody to the highly conserved cyclin binding PSTAIR helix (Fig. 3d). In addition, cyclin A was not detected. Thus, our coimmunoprecipitations appear to be quite specific, and they support the notion that MRN does not associate globally with CDK–cyclins. We interpret the minimal impact on CtIP levels inCDK2−/− cells (Fig. 2d) to indicate that an alternative kinase acts without Mre11 interaction. However, we cannot rule out the pos¬sibility that either weak interaction occurs below our detection limit or an uncharacterized kinase interacts that is not recognized by the PSTAIRE antibody interacts with Mre11.
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