Fig 2: LH signals to LHR, enhancing N-WASP phosphorylation. (A) T-47D cells were treated for different times (0–60 min) with LH (5 mIU/ml). The total cell amounts of wild-type N-WASP and phosphorylated N-WASP (p-N-WASPS484/5) are shown by western blot analysis. (B) Phospho-N-WASP densitometry values were adjusted to N-WASP intensity and then normalized to the control sample. *P < 0.05 vs. control. (C) T-47D BC cells were treated with LH (5 mIU/ml) for 20 min. Cells were stained with phospho-N-WASPS484/5 linked to fluorescein isothiocyanate (FITC, green), actin fibers with Texas Red phalloidin (red), and nuclei were counterstained with DAPI. Yellow arrows indicate actinic cytoskeleton reorganization to the periphery of the cell membrane. White arrows indicate membrane-localized p-N-WASP. (D) T-47D BC cells were transfected with shRNA vs. LHR or with vehicle, and protein analyses for LHR, N-WASP, and phospho-N-WASPS484/855 were performed on cell lysates with or without treatment for 20 min with LH. The total cell amounts of wild-type LHR, N-WASP, and phospho-N-WASPS484/485 are shown by western blot. (E,F) LHR and phospho-N-WASP densitometry values were adjusted to N-WASP intensity and then normalized to the control sample. The results are expressed as the mean ± SD of the measurements. *P < 0.05 vs. CON, control. All experiments were performed in triplicate; representative images are shown.

Fig 3: 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.

Fig 4: WASp interacts with DGKa through the WH1 domain while N-WASp does not bind HEK 293-T cells were co-transfected with the indicated plasmids. 48 hours later, proteins were immunoprecipitated with anti-FLAG antibody and subsequently analysed by western blotting.(A) WASP constructs used with domains and tags evidenced. (B) Interaction between myc-DGKa and FLAG-WASp or FLAG-WASp deletion mutants (FLAG-WASp K232* and FLAG-WASp Q297*) and the corresponding total cell lysates. (C) Interaction between FLAG-WASp or myc-FLAG N-WASp with myc-DGKa WT and the corresponding total cell lysates.