1. Effect of expressing an activated mutant (Rac V12) and an inactivated mutant (Rac N17) of Rac on PTHRP gene expression levels and on JNK activation.
Effect of expressing an activated mutant (Rac V12) and an inactivated mutant (Rac N17) of Rac on PTHRP gene expression levels and on JNK activation. H-500 cells were grown to semiconfluence and transiently transfected with either 10 μg of Rac V12 or Rac N17 constructs as described in“ Materials and Methods.” After transfection, cells were cultured for 24 h in serum-free media. Untransfected and Rac N17-transfected cells were then stimulated with 10% FBS for 2 h. A, 15 μg of total cellular RNA were extracted from control and transfected cells and electrophoresed on a 1.1% agarose formaldehyde gel. After RNA transfer to nylon membranes, the filters were probed with [32P]PTHRP cDNA or with a 32P-labeled 18 S RNA probe as described in “Materials and Methods.” All blots were quantified by densitometric scanning (bottom). Results represent triplicate determinations; bars, SD. ∗, significant difference from controls (P < 0.05). B, H-500 cells cultured under serum-free conditions (CH) or transfected with Rac V12 plasmid were lysed. Alternatively, after 10 min of serum stimulation, untransfected (FBS) and Rac N17-transfected cells were lysed, and JNK-1 protein was immunoprecipitated. Enzymatic activity was determined by immune complex kinase assay using GST-jun fusion protein as substrate as described in “Materials and Methods.” The results are representative of three different experiments and are plotted as fold increase over unstimulated activity. Bars, SD.
Fasika Aklilu et al. Cancer Res 2000;60:
©2000 by American Association for Cancer Research