Active cdc42 detection kit

Active Cdc42 level was detected by using Active Cdc42 Detection Kit (Cell Signaling Technologies, #8819). Briefly, GST-PAK1-PBD fusion proteins were used to bind GTP-bound Cdc42, which was then immunoprecipitated with glutathione resin and detected by western blotting.

The active Cdc42 Detection Kit (Cell Signaling) was used according to the manufacturer's instructions. Briefly, GTP-bound Cdc42 was isolated with glutathione agarose beads bound to the p21-binding domain of PAK1 via a GST tag, and then identified by immunoblotting of eluted proteins with an antibody for Cdc42.

Pak2^+/+ and Pak2^−/− Schwann cells were plated onto poly-L-lysine (PLL)-coated 100 mm plates and grown in Dulbecco’s modified Eagle medium (DMEM)/F12 supplemented with 10% fetal bovine serum (FBS) until they reached 80% confluency. To induce serum and mitogen starvation, cells were washed twice with pre-warmed Hanks’ Balanced Salt Solution (HBSS) and incubated in DMEM/F12 containing 0.5% FBS (starvation medium) for 20 h. After the incubation, three replicates of each plate were treated for 1 h at 37°C and 5% CO₂ using either starvation medium with a vehicle (PBS plus 0.02% FBS; mock medium), 50 ng/ml Nrg1, or 2 μM PrP^C peptide.
Following the stimulation period, cells were washed twice with PBS containing phosphatase inhibitors (Cat. No. 5872, Cell Signaling) and lysed with 1× lysis buffer. Total protein concentration was determined using the BCA assay (Prod. No. 23225, Thermo Scientific). The active GTPase pull-down assay was performed according to the manufacturer’s instructions for the active RAC1 detection kit (Cat. No. 8815, Cell Signaling) and active CDC42 detection kit (Cat. No. 8819, Cell Signaling), with samples treated with GTPγS (positive control) and GDP (negative control). The immunoprecipitated materials and total protein samples were then prepared for immunoblot analysis.