Figure 1
From: Feedback inhibition by RALT controls signal output by the ErbB network
Biological analysis of RALT signalling in mitogenic programs activated by PDGF and bFGF. NIH-EGFR/ErbB-2 cells were seeded in 24-well plates and infected with either Pinco or Pinco–RALT retrovirus stocks. Infection efficiency, as monitored by FACS assessment of GFP-positive cells was >95%. (a) Following infection, cells were made quiescent by 24 h serum deprivation and subsequently challenged with 5 ng/ml EGF for the indicated time. Cell lysates were subjected to immunoblot analysis with either the S1 antiserum that recognizes mouse, rat and human RALT species or MoAb 19C5/4 specific for the ectopically expressed rat RALT species. (b) Following infection, cells were switched to either serum-free medium (SFM) or SFM containing escalating concentrations of EGF (0.3, 1 and 5 ng/ml), bFGF (0.3, 1 and 5 ng/ml) or PDGF (0.2, 2 and 10 ng/ml). Assays were performed in quadruplicate wells. After 44 h, cultures were labelled for 4 h with 1 μCi/ml [3H]methyl-thymidine and cell proliferation measured as fold increase of TCA-precipitated radioactivity in growth factor-treated wells versus control cultures. The s.d. was <15%. Data are representative of one out of three experiments that gave similar results. White bars indicate Pinco cells, gray bars indicate Pinco–RALT cells
