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. 2000 Dec 1;106(11):1399–1407. doi: 10.1172/JCI10536

Figure 3.

Figure 3

NEP enzyme activity inhibits the formation of cSrc and FAK protein complexes. (a) Upper panel: 500 μg of PC total cell lysates were immunoprecipitated with anti-cSrc antibody SRC-2, separated by SDS-PAGE, transferred to nitrocellulose and Western blotted with anti-FAK antibody C20. (a) Lower panel: The same blot was stripped and reprobed with mAb B12 for cSrc protein. (b) Cell lysates derived from LNCaP cells cultured in media without FCS (lane 1), 10 nM ET-1 for 20 minutes (lane 2), 10 nM CGS24592 for 2 hours (lane 3) or CGS24592 followed by ET-1 (lane 4) were analyzed as described in a. (c) (left) TSU-Pr1 cells were cultured in serum-free media (lane 1) with the addition of 10 nM bombesin for 20 minutes (lane 2), or 10 μM of Src kinase inhibitor PP2 for 30 minutes prior to the addition bombesin (lane 3). (right) TSU-Pr1 cells were cultured in media containing 10% FCS (lane 4), 10 μM of PP2 (lane 5), or 50 μg/ml of rNEP for 2 hours prior to PP2. Cells were lysed and analyzed as described in Figure 1b. (d) WT-5, TN-12, and M-22 cells were cultured with (+) and without (–) 1 μg tetracycline for 48 hours (with the addition of 100 nM CGS24592 in lanes 2 and 4). Cells were lysed and analyzed as described in a.