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Knockdown of Arf6 or FE65 or transfection of the FE65-binding domain recovers mutated Sema5A-induced excessive Rac1 activation. ( A, B ) Cells stably harboring wild type Sema5A (WT) or Sema5A p.Arg676Cys (R676C) were transfected with plasmids encoding gRNA for Arf6 (gArf6) or FE65 (gFe65) or luciferase (gControl) plus Cas13 or FE65-binding domain (FBD) or its empty vector (vector) and cultured for 2 days with (+) or without (-) differentiation induction. Cell lysates were analyzed using <t>a</t> <t>G-LISA</t> <t>GTPase</t> kit to assay the amounts of GTP-bound Rac1 or Cdc42 (** p < 0.01; n = 3).
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Knockdown of Arf6 or FE65 or transfection of the FE65-binding domain recovers mutated Sema5A-induced excessive Rac1 activation. ( A, B ) Cells stably harboring wild type Sema5A (WT) or Sema5A p.Arg676Cys (R676C) were transfected with plasmids encoding gRNA for Arf6 (gArf6) or FE65 (gFe65) or luciferase (gControl) plus Cas13 or FE65-binding domain (FBD) or its empty vector (vector) and cultured for 2 days with (+) or without (-) differentiation induction. Cell lysates were analyzed using a G-LISA GTPase kit to assay the amounts of GTP-bound Rac1 or Cdc42 (** p < 0.01; n = 3).

Journal: Scientific Reports

Article Title: Autism spectrum disorder-associated Sema5A p.Arg676Cys drives Arf6/FE65 signaling and aberrant cell morphogenesis

doi: 10.1038/s41598-026-39722-x

Figure Lengend Snippet: Knockdown of Arf6 or FE65 or transfection of the FE65-binding domain recovers mutated Sema5A-induced excessive Rac1 activation. ( A, B ) Cells stably harboring wild type Sema5A (WT) or Sema5A p.Arg676Cys (R676C) were transfected with plasmids encoding gRNA for Arf6 (gArf6) or FE65 (gFe65) or luciferase (gControl) plus Cas13 or FE65-binding domain (FBD) or its empty vector (vector) and cultured for 2 days with (+) or without (-) differentiation induction. Cell lysates were analyzed using a G-LISA GTPase kit to assay the amounts of GTP-bound Rac1 or Cdc42 (** p < 0.01; n = 3).

Article Snippet: Cells were lysed in ice-cold G-LISA cell lysis buffer (G-LISA GTPase kit, Cytoskeleton, Inc., Denver, CO, USA).

Techniques: Knockdown, Transfection, Binding Assay, Activation Assay, Stable Transfection, Luciferase, Plasmid Preparation, Cell Culture