FIG. 1.

Analysis of c-fos gene expression in cell lines harboring ts mutant protein p53Val135. (a) Poly(A)+ RNA was extracted from M1 and LTR6 cells incubated at either 37 or 32°C for 4 h. Aliquots (5 μg) were separated by agarose gel electrophoresis, transferred to a nylon membrane, and probed with a mouse c-fos cDNA probe. The same membrane was subsequently reprobed for GAPDH. Total cellular RNA was extracted from LTR6 (b) or Clone6 (c) cells following incubation at 32°C for the indicated periods. An 8-μg sample of each RNA was subjected to semiquantitative RT-PCR performed with c-fos- and GAPDH-specific primers. The numbers of PCR cycles for c-fos and GAPDH were 26 and 21 (b), and 30 and 21 (c) respectively. (d) Nuclear extracts were prepared from M1, LTR6 and Clone6 cells incubated at either 37 or 32°C for 4 h. Equal amounts of protein (100 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and probed with an anti-c-Fos antibody. Total cellular RNA was extracted from MCO1-cG9 or MCO1 cells following either incubation at 32°C for the indicated periods (e) or starvation for 24 h in 0.1% FCS, followed by incubation in 20% FCS for the indicated periods (f). A 5-μg sample of each RNA was subjected to semiquantitative RT-PCR as described above. The numbers of PCR cycles were 30 and 20 (e) and 26 and 20 (f) for c-fos and GAPDH, respectively.