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. 2017 Dec 26;115(2):E190–E199. doi: 10.1073/pnas.1708584115

Fig. 7.

Fig. 7.

Deletion of LARG impairs RSK2 activation of RhoA and cell migration and invasion. (A and B) LARG was deleted in U87MG cells by CRISPR/Cas9-targeted deletion as described in Materials and Methods. LARG-deleted cells and nonspecific CRISPR/Cas9-treated control cells expressing HA-RhoA-WT were stimulated with FBS (10%, 10 min), EGF (100 ng/mL, 5 min) (A), or transfected with Flag-EV, Flag-RSK2-T577E, or Y707A (B). GTP-RhoA was retrieved using GST-rhotekin-RBD, and GTP-RhoA levels were determined by immunoblotting with anti-HA antibody as described above. Expression levels of HA-RhoA, LARG, and Flag-RSK2 were determined by immunoblotting. (C) RSK2-induced migration and invasion was impaired in LARG-deleted U87MG cells. CRISPR/Cas9-mediated nonspecific control or LARG-deleted cells were transfected with Flag-EV, RSK2-T577E, and Y707A. Transfected cells were enriched by G418 selection and serum starved for 24 h before migration and invasion assays. Cell migration (Upper) and invasion (Middle) were assessed by Transwell assays. Refer to Dataset S1D for all P values. Expression levels of RSK2 and LARG proteins are shown (Lower). *P < 0.05, **P < 0.01, ***P < 0.001.