Figure 4

β-AR mediated activation of the MAPK pathway. UROtsa cells were incubated with 100 nM isoproterenol or 300 nM LPS for the indicated times, and cell lysates were immunoblotted with anti-pERK to determine the receptor mediated phosphorylation state of ERK or anti-ERK2 to establish the total amount of ERK2 in the samples. As per manufacturer's literature, pERK antibody binds phorphorylated ERK1 and ERK2 in two bands found at 44 and 42 kD, respectively. The ERK2 antibody only binds ERK2 at 42 kD. Peak levels of pERK were observed 5 min after addition of isoproterenol with no significant changes in the total cell lysate levels of ERK2. Densitometric analysis demonstrated a significant 2.4 ± 0.4 fold increase in pERK levels when compared to basal. Similarly, LPS treatment also increased ERK phosphorylation over basal within 5 min without a change in ERK2 production. Values are presented as the mean ± S.E. and the autoradiographs are representative of n = 3–10 independent UROtsa cell treatments.