FIGURES AND TABLES
FROM:
Mutant K-ras oncogene regulates steroidogenesis of normal human adrenocortical cells by the RAF-MEK-MAPK pathway
C-H Wu, Y-F Chen, J-Y Wang, M-C Hsieh, C-S Yeh, S-T Lian, S-J Shin and S-R Lin
BACK TO ARTICLE
Figure 1.
Time course of RasGTP formation after treatment with IPTG. The normal human adrenocortical cells transfected with pK568MRSV plasmid were induced with 2.5 mM IPTG. Then the active RasGTP form was precipitated by GST-RBD of Raf-1 fusion protein and immunoblotted as described in Materials and Methods. The RasGTP levels were detected at 0, 1, 6, 12, 24, 36 and 48 h after IPTG induction. Upper panels show the amount of RasGTP; lower panels show total amount of Ras in 10% of the extract. The amount of RasGTP was increased with the elongation of IPTG induction. Plus (+) indicates when present; minus (-) indicates when absent.
Full figure and legend (5K)
Figure 2.
Time course of the phosphorylation of c-Raf1 and MAPK in pK568MRSV transfected adrenocortical cells after treatment with IPTG. Crude protein extract from transfected cells was electrophoresised and transfered to PVDF as described in Materials and Methods. And then, it was immunobloted with monoclonal antibodies specific to phosphorylated c-Raf1 (p-Raf-1), phosphorylated MAPK and control c-Raf-1 and MAPK, respectively. The amounts of phosphorylated c-Raf-1 (p-Raf-1) and phosphorylated MAPK; and control MAPK and c-Raf-1 were detected at 0, 1, 6, 12, 24, 36 and 48 h after IPTG induction. The amounts of phosphorylated c-Raf-1 and MAPK were increased with the elongation of IPTG treatment. Plus (+) indicates when present; minus (-) indicates when absent.
Full figure and legend (9K)
Figure 3.
The effect of PD098059 pretreatment and the time course of MEK activity in pK568MRSV transfected adrenocortical cells after treatment with IPTG. Kinase activity was measured using myelin basic protein (MBP) as a substrate as described in Materials and Methods and significantly increased at 6, 12, 24 and 36 h after IPTG induction. *The 36* lane represented the pK568MRSV transfected cells treated with 10 
M PD098059 for 45 min before the addition of 2.5 mM IPTG and then incubation for 36 h in the presence of PD098059 and IPTG. With pretreatment of PD098059, the MEK activity was obviously inhibited in pK568MRSV transfected adrenocortical cells after treatment with IPTG. Plus (+) indicates when present; minus (-) indicates when absent.
Figure 4.
Effect of PD098059 on cortisol production or P450scc, P450c17 and 3HSD mRNA in mutant K-ras gene transfected human adrenocortical cells. pK568MRSV or pOPRSVI transfected adrenocortical cells were treated with IPTG for 1, 6, 12, 24 and 36 h. Then the total RNA was extracted from the treated cells for Northern blot analysis. The histogram revealed the densitometry data of Northern blot of p450scc, p450c17 and 3HSD at every time point. The data indicates that their mRNA levels increased with incubation time, however, the increase was blocked by PD69859 as shown in lane 36*. Cortisol level in culture medium was detected by EIA and was found to increase with the elongation of IPTG induction. And the elevated level of cortisol was blocked shown in (B) the 36* column. (B) *The 36* lane represented the pK568MRSV transfected cells treated with 10 M PD098059 for 45 min before the addition of 2.5 mM IPTG and then incubation for 36 h in the presence of PD098059 and IPTG. Plus (+) indicates when present; minus (-) indicates when absent.
