C. Cai et al. Figure 6. KHDC1 proteins associate with mCPEB1. (A) Flag-KHDC1A and Flag- KHDC1B coimmunoprecipitate with HA- mCPEB1 expressed in Xenopus embryos or in 293T cells. (B) The interaction between KHDC1B and mCPEB1 is independent of RNA. (C) Full-length mCPEB1 and the dele- tion constructs used to test for interaction with KHDC1B. The PEST domain is a motif that regulates protein stability. The RRM (RNA recognition motif) and ZF (zinc ﬁnger) domains control RNA binding. (D) HA- mCPEB1 and HA-mCPEB1-Cter coimmuno- precipitate with Flag-KHDC1B when coex- pressed in 293T cells. HA-CPEB-Nter did not interact with KHDC1B in this assay. * in the input indicates nonspeciﬁc signal. For input, 5% of the total lysate was loaded. In addition to exhibiting cell cycle– dependent changes in protein levels, KHDC1B binding partners may also be cell cycle– dependent. eIF4E is a known component of the CPEB complex and is a target of the translational repressor Maskin (Stebbins-Boaz et al., 1999). KHDC1B was found to weakly associate with the translational regulator eIF4E; however, when cells were synchronized at metaphase by nocodazole, the interaction was strengthened (Figure 8C). These data demonstrate that, like Maskin, KHDC1B may associate with eIF4E under speciﬁc conditions. DISCUSSION In this study, we describe the expression pattern and activity of two related KH-domain proteins. The founding member of this family, Ndg1/Khdc1a was originally identiﬁed in T-cells in a screen for target genes of the orphan nuclear receptor NUR77 (Rajpal et al., 2003). Pierre et al. (2007) identiﬁed two additional family members in mice and proposed that the KHDC1 family 3144 was part of a larger superfamily of RNA binding proteins expressed in oocytes of eutherian mammals. We have con- ﬁrmed that both Khdc1a and Khdc1b are expressed in mouse oocytes but ﬁnd that Khdc1a has a wider expression proﬁle with mRNA detected in lung, brain, testes, and ovaries. KHDC1A protein was detected in liver, kidney, intestine, tes- tes, and ovaries. In addition to the expected sized protein, we detected a smaller-molecular-weight isoform in testes and late stage oocytes. We speculate that this smaller-weight form might represent a splice variant or cleavage product speciﬁc to germ cells. We do not know whether KHDC1B protein is expressed because we currently lack an antibody speciﬁc for this family member. The KHDC1 proteins probably have both nuclear and cyto- plasmic functions. In growing follicles the KHDC1 proteins are predominately cytoplasmic, but we did observe intense KHDC1 staining within the nucleus of some oocytes. This probably represents KHDC1B protein considering that 1B was the only form to exhibit nuclear localization when transiently Molecular Biology of the Cell