Fig. 1. Characterisation of the chicken INK4b and ARF gene products. (A) CEFs (32-38 PDs) were infected with retroviruses encoding 2xHA-tagged versions of chicken ARF and INK4b, or empty vector control (Vec). Pools of drug-resistant cells were analysed for expression of HA-tagged proteins (2HA-ARF, 2HA-INK4b), p53 and p21^CIP1. Actin was used as a loading control. (B) At 8 days post-infection, viable cell numbers were compared by staining the cells with Crystal Violet. The results represent the OD at 590 nm averaged from three independent cell pools. In a separate experiment, equivalent cell pools were labelled with BrdU for 1 hour and the proportion of BrdU-positive cells determined by immunohistochemistry. (C) Samples of lysates from CEFs expressing 2xHA-tagged chicken INK4b or ARF (as in panel A) were immunoblotted with polyclonal antisera raised against ARF (SK8) or INK4b (SK14) peptides. The asterisk identifies the background band detected with the SK8 antibody, as discussed in the text. (D) CEFs (36 PDs) were infected with a retrovirus encoding human E2F1, or empty vector, and analysed for expression of endogenous ARF and INK4b by immunoblotting with the SK8 and SK14 antisera respectively. The levels of p53 were also assessed using the HP64 monoclonal antibody. Actin served as loading control. The asterisk identifies the background band detected with the SK8 antibody, as discussed in the text.