Our results demonstrate that unlike FAK, Pyk2 does not play a critical role Gefitinib EGFR/HER2 inhibitor during the immediate response to FF-induced mechanotransduction in osteoblasts. We show that Pyk2 deficient osteoblasts, unlike FAK deficient osteoblasts, exhibited appropriate increases in c-Fos and COX-2 protein levels, increases in phosphorylation of ERK and appropriate increases in OPN expression in response to FF. Additionally, we have compared two methods of generating FF and determined that these two methods induce a similar response in both primary calvarial osteoblasts and immortalized calvarial osteoblast clones. Our previous work has clearly demonstrated that FAK is key component of FF- induced mechanotransduction in osteoblasts during short periods of mechanical stimulation. This study further demonstrates the importance of FAK during the response of osteoblasts to short periods of FF, and it supports the idea that FAK functions in a mechanosome signaling complex. Additionally, our data indicates that FAK and Pyk2 do not have redundant functions in osteoblasts leading us to hypothesize that Pyk2 may function during long-term. Perhaps Pyk2 and FAK work in concert to regulate both short and long periods of FF in osteoblasts. We suggest that Pyk2 may still be involved in mechanosome signaling complexes because it shuttles away from the membrane and to the nucleus in response to mechanical stimulation. While we currently do not have data to directly support this hypothesis, we are eager to test the role of Pyk2 during long term mechanical stimulation. While our work indicates that Pyk2 is not involved in the immediate response to FF, we have yet to determine if Pyk2 is involved in mechanically induced osteoblast differentiation, which requires periods of FF over the span of several days. Buckbinder et al. demonstrated an increase in alkaline phosphatase activity and increased bone mineralization in bone marrow cultures from Pyk22/2 mice, which indicates that Pyk2 may be involved in the regulation of differentiating pre-osteoblasts. These data together with the osteopetrotic phenotype of the Pyk22/2 mice suggest that Pyk2 may be involved in regulating mechanically induced osteoblast differentiation in a mechanosome signaling complex, and we are in the process of testing this hypothesis. Prior to this study, we have been unable to examine the role of Pyk2 during long periods of FF over the course of several days because of the limiting abilities of the oscillatory pump/parallel plate flow chamber method. Specifically, this method is very difficult to keep sterile for several days. Additionally, we were concerned that the 300 ml volume of media in the chamber was not adequate for the respiration and nutrient exchange needs of the cells over the course of several hours or days. This led us to test the orbital platform method. The orbital platform method puts to use an orbital platform shaker and 6 well plates instead of the parallel plate chambers and glass slides.