Fig. 5. Rac1 and Cdc42 increased in the plasma membrane after A_1-42 stimulation. (A) Four-day hippocampal neurons, untreated (control) and exposed to A_1-42 fibrils for 4 hours, were fixed after detergent extraction performed under cytoskeleton-stabilizing conditions and double immunostained with Rac1 or Cdc42 and Rhodamine-phalloidin. Both GTPases were recruited to the plasma membrane and were frequently found to colocalize with F-actin after amyloid stimulation (arrowheads). Bar, 20 µm. (B) Embryonic hippocampal cells, untreated and treated for 4 and 24 hours with -amyloid, were lysed and cytoplasmic and membrane fractions obtained. Rho GTPases recruitment to the membrane fraction was analyzed using Rac1 and Cdc42 antibodies and the values were normalized against flotillin as a membrane protein control. Samples from the cytoplasmic fraction were analyzed by immunoblotting using Rac1 and Cdc42, along with actin antibody as a loading control. Significant differences are indicated by asterisks (^*P<0.05, ^**P<0.01; Student's t-test).