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Fig. 1. Downregulation of RhoA-GTP in response to PLXNB1 activation. (A) NIH-3T3 fibroblasts either expressing PLXNB1 or mock transfected, were treated with the ligand Sema4D (5 nM) for the indicated times. RhoA-GTP levels were measured by pull-down with rhotekin-RBD-coated beads, followed by immunoblotting with anti-RhoA antibodies. Expression levels of RhoA and of the exogenous plexin in input lysates are shown below. The amount of pulled-down GTP-RhoA relative to total RhoA levels in each condition is reported at the bottom, based on band intensity quantification. (B) Measurement of RhoA-GTP levels by rhotekin-RBD pull-down, as above, in SKBR3 cells expressing endogenous PLXNB1 and treated with 10 nM Sema4D for the indicated times. Relative amounts of active RhoA were measured as above and are shown at the bottom.
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