Fig. 2. The Ras/ERK signalling cascade affects pol III transcription independently of RB. (A) CCL39-ΔRaf-1:ER cells were arrested by serum starvation for 24 h. RNA was extracted from cells that were left untreated (lane 1) or stimulated either with 10% serum for 24 h (lane 5) or with 1 µM estradiol for 2 h (lane 2), 10 h (lane 3) or 24 h (lane 4). The upper and lower panels show northern blots probed with B2 and ARPP P0 as above. (B) Protein (10 µg) extracted in parallel from the above cells was resolved by SDS–PAGE and then immunoblotted with antibodies against RB phosphorylated at serine 780 (upper panel), cyclin D1 (second panel), TBP (third panel) or actin (lower panel). (C) Templates (500 ng) VA1 (upper panel) and tRNALeu (lower panel) were transcribed using 15 µg of whole-cell extract prepared from 3T3 cells grown continuously in 10% serum in the presence of vehicle (lane 1) or 1 µM U0126 for the times indicated. (D) Whole-cell extracts from the above experiment were resolved by SDS–PAGE and analysed by western blotting with antibodies against RB phosphorylated at serine 780 (upper panel), cyclin D1 (second panel) or actin (lower panel). (E) Wild-type (wt; lanes 1 and 2) or Rb–/–p130–/–p107–/– triple knockout mouse embryo fibroblasts (TKO; lanes 3 and 4) were transfected with pVA1 (0.5 µg, all lanes), pCAT (0.5 µg, all lanes), pCMV vector (3 µg, lanes 1 and 3) or pCMV-RasV12 (3 µg, lanes 2 and 4). VA1 and CAT RNA levels were assayed by primer extension. VA1 RNA levels are shown; CAT levels remained constant (data not shown). (F) VA1 RNA levels are shown; CAT levels remained constant (data not shown). VA1 and CAT levels from (E) were quantified by PhosphorImager as before. Values presented graphically are means and standard deviations for VA1 expression after normalization to the levels of CAT RNA to correct for transfection efficiency. The activities obtained for wt cells transfected with pCMV alone (lane 1) were set at 100 and other values were calculated as a percentage of this.