G lisa

FITC-labeled Aβ42 was purchased from GL Biochem Ltd. (Shanghai, China); TMR-labeled Aβ42, FITC-dextran (MW: 70,000), a marker for the macropinocytosis pathway, FITC-Cholera Toxin B subunit (CTB), a marker of Lipid raft, heparin sodium salt an inhibitor of HSPGs, and methyl-β-cyclodextrin (MβCD) that can disrupt lipid raft, were obtained from Sigma-Aldrich (St. Louis, MO, USA); ethylisopropyl amiloride (EIPA) a blockef of macropinocytosis, SecinH3, an indirect inhibitor of Arf6, Grassofermata (NAV 2729) a direct inhibitor of Arf6, and EHT1864, a Rac1 inhibitor of were purchased from Cayman chemical (Ann Arbor, MI, USA); dynasore (ab120192), a dynamin inhibitor was from Abcam (Cambridge, MA, USA), Alexa Fluor 488 transferrin and phalloidin were obtained from Invitrogen (Carlsbad, CA, USA); wortmannin, a phosphoinositide 3-kinase inhibitor, Dulbecco’s Modified Eagle Medium (DMEM) cell culture medium, phenol red-free Ham’s F12 Medium, 0.25% trypsin-EDTA, and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) were from Fujifilm Wako Pure Chemical Corporation (Osaka, Japan); fetal bovine serum was from COSMO BIO (Tokyo, Japan); and Hoechst 33342 and 4’,6-diamidino-2-phenylindole (DAPI), were from Dojindo (Kumamoto, Japan). G-LISA (ELISA-based GTPase activation assay) kits obtained from Cytoskeleton (Denver, CO, USA) were used to measure the activities of Rac1 and Arf6.

The intracellular amounts of Rac1-GTP and RhoA-GTP were determined using the G proteins-linked assay (G-LISA) (Cytoskeleton, Inc., Denver, CO, USA) according to the manufacturer's instructions. In brief, cells were washed with cold PBS and homogenized gently in ice-cold lysis buffer. A measure of 20 μl was removed for protein quantification to adjust sample concentration to 0.5 mg ml⁻¹. After adding an equal volume of binding buffer, triplicate assays were performed using 1.5 μg protein per well. Samples were incubated for 30 min and then washed three times with washing buffer. Antigen-presenting buffer was added for 2 min before removal; samples were then incubated with 1:250 dilution of anti-Rac1 and anti-RhoA antibodies, respectively, at room temperature for 45 min, washed three times and incubated with secondary antibodies for another 45 min. HRP detection reagent was added and signal was read by measuring absorbance at 490 nm using a microplate spectrometer.

RhoA activity was determined by G-LISA (Cytoskeleton) following manufacturer’s instructions.

NB cells were seeded in 6-well plates. After 24 h cells were transfected with FZD2 and scrambled siRNA. 24 hours after transfection cells were starved for 24 h. Cells were stimulated with 100 ng/ml recombinant Wnt3a and Wnt5a protein for 30 min and Rac1 activity was then measured with G-LISA (colorimetric format, Cytoskeleton, Denver, CO) according to the manufacturer's protocol.