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. 2021 Feb 5;8:630147. doi: 10.3389/fcell.2020.630147

FIGURE 4.

FIGURE 4

LH stimulates the Src/FAK/paxillin/cortactin/N-WASP and Arp2/3 complexes. (A,B) BC cells were exposed to LH (5 mIU/ml) for 20 min in the presence or absence of the c-Src kinase inhibitor PP2 (10 μM). Cell protein extracts were immunoprecipitated with an antibody vs. FAK (A) and cortactin (B). The immunoprecipitates (IPs) were assayed for co-immunoprecipitation vs. FAK, p-FAK, cortactin, and the Arp3 subunit. The membranes were re-blotted for the immunoprecipitated protein to show equal input. (C) Cells were incubated in the presence of LH (5 mIU/ml) for 20 min with or without PP2 (10 μM), FAK (1 μM), and/or dominant negative constructs vs. cortactin (cortactin3YF). The total cell amounts of wild-type c-Src, FAK, paxillin, cortactin, and N-WASP or p-SrcY416, p-FAKY397, p-paxillinY118, p-cortactinY466, and p-N-WASPS484/485, respectively, are shown by western blot. (D) BC cells were transfected with the dominant negative constructs of cortactin (cortactin3YF), the specific inhibitor of N-WASP (Wiskostatin, 10 μM), and/or the inhibitor of the Arp2/3 complex (CK-666, 4 μM) and incubated in the presence of LH (5 mIU/ml) for 20 min. Phospho-N-WASPS484/485 and phospho-Arp2T237 were assayed by western blot analysis. (E,F) T-47D cells were exposed to LH (5 mIU/ml, 20 min) in the presence or absence of CK-666. Cell protein extracts were immunoprecipitated (IP) with an antibody vs. Arp2. IP was assayed for co-immunoprecipitation vs. p-Tyr. The membranes were re-blotted for the immunoprecipitated protein vs. Arp2 to show equal input. All experiments were performed in triplicate with consistent results; representative images are shown. *P < 0.05 vs. CON, control.