Biochemistry in p185^BCR-ABL-transformed cell lines. (A) Expression levels of Cdk4, Cdk6, and INK family members in 3 individually derived cell lines of each genotype as indicated. (B) Phosphorylation of Rb at Ser780 or Ser807/811 is significantly increased in Cdk4^R/R; Cdk6^R/R as compared with single-mutant or control cultures. Depicted is the summary from 3 independent western blots using 3 or 4 independent p185BCR-ABL–transformed cell lines. *P < .05; **P < .01; ***P < .001. One-way ANOVA including Bonferroni’s multiple comparison test. (C) Representative blots of Rb phosphorylation in the indicated residues in 4 independent clones per genotype. (D) Protein levels of p16^INK4a, Cdk4, and Cdk6 after immunoprecipitation (IP) with antibodies against Cdk4. I and SN indicate the input lysate or supernatant after IP. The relative enrichment of p16^INK4a complexed with Cdk4 calculated from 2 individual IP experiments is shown in the histogram. Student t test; ***P < .001. (E) Protein levels of p16^INK4a, Cdk4, and Cdk6 after IP with antibodies against Cdk6 (IP). I and SN indicate the input lysate or supernatant after IP. The histogram depicts the relative enrichment of p16^INK4a complexed with Cdk6 calculated from 3 individual IP experiments. Student t test; *P < .05. (F) Protein levels of p16^INK4a, Cdk4, and Cdk6 after IP with antibodies against p16^INK4a. I and SN indicate the input lysate or supernatant after IP. The histogram shows the quantification (relative to wild-type cells) of the amount of p16^INK4a bound to Cdk4 or Cdk6 in the different clones. (G) Overexpression of wild-type Cdk6 (Cdk6-wt) reduces the levels of Cdk4-bound p16 in Cdk4^+/+; Cdk6^R/R cells. In all of these assays, Hsc70 was used as a loading control.