Activation of ERK 1/2 in response to 15 min OFSS is enhanced in Nmp4 KO osteoblasts compared to WT controls. (A) Representative immunoblot of total cellular protein from Nmp4 and WT osteoblasts maintained in static culture or subjected to OFSS for 15 min. Blots were probed either with antibody specific for phosphorylated ERK 1/2 or for total ERK 1/2 protein. (B) Quantification of immunoblots illustrated in (A), n=3. The static vs. 15 min OFSS experiment was repeated three times with comparable results. *p<0.05 vs static; #p<0.05 vs WT flow. (C) Representative time course of ERK 1/2 phosphorylation at 5, 15, 30 and 60 min of OFSS. Although phosphorylation of ERK 1/2 in Nmp4 KO osteoblasts was consistently elevated above WT osteoblasts in response to OFSS at all time points, and was significantly higher at 15 min, statistically significant differences could not be measured at 5, 30 or 60 min (not shown).