Fig. 1. Protein levels of cdk4 are induced in tissues of C/EBPα-knockout mice. (A) Western analysis of cdk4 expression in liver. Nuclear extracts or cytoplasm from newborn wild-type (WT), heterozygous (H) or C/EBPα-knockout (KO) livers were loaded on a denaturing gel and probed with antibodies to cdk4. The membranes were stripped and re-probed with antibodies to β-actin to verify protein loading. C, control extract containing high levels of cdk4. Protein levels of cdk4 were calculated as their ratio to β-actin. Summaries of three to five experiments are shown below as bar graphs. (B) Northern analysis of cdk4 mRNA in livers from wild-type, heterozygous and C/EBPα-knockout littermates. Results from two animals of each genotype are shown. The membrane was re-probed with 18S rRNA probe. The intensity of signals was determined using phosphoimaging and the levels of cdk4 mRNA were calculated as a ratio to 18S rRNA. The bar graph show a summary of data with five animals per genotype. (C) Expression of cdk4 in brown fat (BF), lung and kidney tissue. Western blotting with nuclear extracts was performed as described in Materials and methods. Results with two or three animals of each genotype are shown. The table shows the induction of cdk4 protein in tissues from C/EBPα-knockout animals as ratio to the level of cdk4 in wild-type animals. Expression of C/EBPα in these tissues is shown based on published observations (Birkenmeier et al., 1989).