Figure 2.

Status of cell cycle regulatory components at the permissive and non-permissive temperatures. (A) SVts8 cells cultured in the presence and absence of PD0332991 were switched from 34°C to 39°C for 6 d and cell lysates were fractionated by SDS-PAGE and immunoblotted with antibodies that detect specific phosphorylation events on pRb. MEK was used as a loading control. (B) Similar analysis of BJ-TERT-tsLT cells 6 d after temperature shift with antibodies against cyclin A (CCNA), p53, p21^CIP1 and CDK4. (C) Effects of temperature shift on p21^CIP1 and p16^INK4a levels in SVts8 cells. (D) SVts8 cells at 34°C were infected with a retrovirus encoding shRNA against p21^CIP1 (+) or an empty vector control (−). After antibiotic selection, cells were replated and grown at 34°C or 39°C for 48 h. Equivalent amounts of cell lysate were either analyzed directly by immunoblotting for p21^CIP1, p16^INK4a and MEK or immunoprecipitated with an antibody against CDK2 and assayed for their ability to phosphorylate GST-pRb in the presence of [γ-³²P]ATP. (E) Phosphorylation status of pRb in SVts8 cells transduced with p21^CIP1 or control shRNA. Equivalent amounts of cell lysate were analyzed directly by immunoblotting with the indicated antibodies. (F) SVts8 cells transduced with p21^CIP1-specific shRNA continue to grow at 39°C for at least 7 d The plots record cell numbers relative to those before the temperature shift.