Cdc42

Antibodies used in this study included the polyclonal rabbit antibodies against NR2B (#4207, Cell Signaling Technology, Beverly, MA, USA), NR1 (#5704, Cell Signaling Technology), GluA1 (#13185, Cell Signaling Technology), GluA2 (#13607, Cell Signaling Technology), GluA3 (#3437, Cell Signaling Technology), GluA4 (#8070, Cell Signaling Technology), PSD-95 (#3450, Cell Signaling Technology), phospho-Cofilin (#3311, Cell Signaling Technology), Cofilin (#5175, Cell Signaling Technology), Arp3 (#4738, Cell Signaling Technology), NR2A (#05-901R, Millipore, Merck KGaA, Darmstadt, Germany), NR2C (#OPA1-04020, Millipore), NR2D (#PA5-87624, Millipore), AKAP150 (#07-210, Millipore), HMGB1 (#10829-1-AP, Proteintech, Rosemont, IL, USA), Calnexin (#10427-2-AP, Proteintech), Synaptophysin (#17785-1-AP, Proteintech), Iba-1 (#019-19741, Wako Chemicals, Osaka, Japan), and the monoclonal mouse antibodies against NR2A (#MA5-27692, Invitrogen, Carlsbad, CA, USA), NR2B (#MA1-2014, Invitrogen), β-actin (#66009-1-Ig, Proteintech), GAPDH (#60004-1-Ig, Proteintech), Syntaxin (#66437-1-Ig, Proteintech), NeuN (#MAB377, Millipore), RhoA (#ARH04, Cytoskeleton Inc., Denver, CO, USA), Rac1 (#ARC03, Cytoskeleton), Cdc42 (#ACD03, Cytoskeleton), and Rac1-GTP (#26903, Neweast Biosciences, Wuhan, China). Pharmacological agents included glycyrrhizin (#50531, Sigma, St. Louis, MO, USA).

The proteins isolated from cells using RIPA buffer and PMSF were centrifuged at 12,000 rpm for 15 min at 4 ºC, boiled for 5 min. Equal levels of proteins were separated into 10% or 12% SDS-PAGE, a 6% SDS-PAGE used to detect Trio, and then transferred into PVDF membranes, blocked with 5% non-fat milk for 2 h and then incubated with primary antibodies against the following epitopes: Trio (1:200, Abcam, USA), Myh9 (1:100, Proteintech), Rac1 (1:500, Cytoskeleton), Cdc42 (1:250, Cytoskeleton), PAX7 (1:1000, Proteintech), SNAI2 (1:1000, Proteintech), FOXD3 (1:1000, R&D Systems), SOX9 (1:1000, Cell Signaling Technology), GAPDH (1:8000, Bioworld), Histone H3 (1:1000, Cell Signaling Technology) overnight at 4 ºC. The membranes were washed with Tris buffered saline (TBST) 3 times and then incubated with secondary antibody (1:8000, KPL, USA) for 1 h at room temperature, washed with TBST 3 times again. The enhanced chemiluminescence (Thermo Fisher Scientific, Rockford, IL) was used to visualize proteins. Proteins were imaged with Chemiluminescence gel imaging system (Tonon 5200) and semi quantified by Image J software.

Stadie-Riggs tissue slicer was purchased from Thomas Scientific (Swedesboro, NJ); RhoA, Cdc42 and filamentous actin (F-actin) to globular actin (G-actin) (F/G-actin) ratio assay kits were purchased from Cytoskeleton (Denver, CO); the Rho kinase inhibitor Y27632 and the Arp2/3 complex inhibitor CK666 were purchased from EMD Millipore (Billerica, MA); FAK inhibitor FP 573228 and the actin polymerization inhibitors latrunculin A, and cytochalasin D were purchased from Tocris Bioscience (Bristol, UK); All PCR reagents, RNA isolation kit, TURBO DNase, SuperScript II, Lipofectamine 2000 Transfection Reagent as well as phospho-Pyk2(Tyr402) and phospho-paxillin(Tyr118) antibodies were purchased from Thermo Fisher Scientific (Rockford, IL). Anti-Arp2/3, N-WASP antibodies were purchased from Abcam (Cambridge, UK); Cdc42 antibody, phospho-FAK (pY397), and paxillin antibodies were purchased from BD Biosciences; and Cool1/β-Pix, Cool2/α-Pix, DOCK 180, and FAK antibodies were purchased from Cell Signaling Technology (Danvers, MA). Western blot supplies were purchased from Bio-Rad Laboratories (Hercules, CA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO)

Protein extracts from tissue or transfected HEK293 cells were prepared with RAPI or 2×SDS sample buffers according to the standard protocols. Protein A Dynabeads (Invitrogen, Cat.# 1000D) were used for pulling down anti-GFP antibody (Rabbit anti-GFP , Torrey Pines Biolabs, Inc, TP401) associated Prickle1 protein complex at 4C overnight. Proteins were dissociated from the beads by heating in 2×SDS sample buffer at 98C for 5 min, then run a western blot and probed with anti-Ubiquitin antibody and anti-GFP antibody.
Custom-made antibodies for Prickle1 were generated in our laboratory by immunizing rabbits with bacterially expressed recombinant proteins corresponding to aa 344–464 (Pk1C) and aa 711–849 (Pk1N), respectively. Antisera were affinity purified and were used at 1:1000 dilutions for Western blots. Mouse anti-γ-tubulin (Sigma, T-6557) was used at 1:3000; Rabbit anti-Wnt5a was used at 1:1000 (Abcam, ab72583); RhoA, Cdc42 and Rac1 (Cytoskeleton, Inc) were used at 1:1000; rabbit anti-Dvl2 (Chemicon, AB5972; Cell Signaling, Cat# 3216) and rabbit anti-GFP were used at 1:2000; mouse anti-Ubiquitin antibody (ab7254) was used at 1:1000.