MSC were activated by addition of 10 µM (left panel) and 1.5 mM SNP (right panel) for different lengths of time as indicated, and phosphorylation of intracellular signaling proteins was recorded. A transient phosphorylation of c-RAF was observed by low and high dosages of SNP. MEK1/2 and ERK1/2 were found in a phosphorylated stage even prior to stimulation (t=0), whereas JNK and p53 proteins were not. Addition of 1.5 mM SNP resulted in a substantial phosphorylation signal for MEK-1/2 and JNK eight and twelve hours after stimulation. Low SNP failed to do so. The p53 protein was modified by phosphorylation of serine at position 15 after addition of 1.5 mM SNP, but not by 10 µM SNP. Reprobing of the membrane with anti-β-actin served as the loading control.
