Anti rhoa

Cells were lysed in modified radioimmunoprecipitation assay buffer, and lysates resolved using SDS-PAGE. Gels were transferred to polyvinylidene fluoride membrane, followed by blotting with primary antibodies, including anti-Swiprosin-1 (Imgenex, San Diego, CA), anti-Rac1 (BD Biosciences, San Jose, CA), anti-RhoA (Cytoskeleton, Denver, CO), anti-Myc (Cell Signaling Technology, Boston, MA), anti-Cdc42, anti-Swiprosin-2, anti-pEGFR, anti-EGFR and anti-Actin (Santa Cruz Biotechnology, Dallas, TX) antibodies.

Anti-Atg5 and anti-LARG antibodies were purchased from Sigma-Aldrich (St. Louis, MO). Anti-paxillin, anti-p62, anti-actin, and anti-tubulin antibodies were purchased from BD Biosciences (San Jose, CA), MBL (Aichi, Japan), Millipore (Billerica, MA), and Invitrogen (Carlsbad, CA), respectively. Anti-GEF-H1, anti-RhoB, and anti-RhoC antibodies were from Cell Signaling Technologies (Danvers, MA). Anti-RhoA and anti-Rac1 antibodies were from Cytoskeleton Inc. (Denver, CO). Anti-LC3, anti-phosho FAK and anti-GAPDH were purcharsed from NanoTools (Teningen, Germany), Abcam (Cambridge, UK) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. Bafilomycin A1 was obtained from Sigma-Aldrich. Rho inhibitor 1 was obtained from Cytoskeleton Inc. and Chemdea LLC. (Ridgewood, NJ), respectively. All other chemicals were from Wako Co. (Osaka, Japan).

Antibodies with working dilution, company source, and catalog number are listed as below: anti-CD44 (1:200 for IF or 1:1000 for WB; Abcam, #ab119863), anti-Hermes-1 (1:100; Bioxcell, #BE0262), anti-N-cadherin (1:200 for IF or 1:1000 for WB; BD Bioscience, #610921), anti-β-catenin (1:300 for IF or 1:1500 for WB; BD Biosciences, #610154), anti-E-cadherin (1:300 for IF or 1:1500 for WB; BD Biosciences, #610182), anti-γ-catenin (1:200 for IF or 1:1000 for WB; BD Biosciences, #610253), anti-RhoA (1:200 for IF or 1:1000 for WB; Cytoskeleton, #ARH03), anti-Rac1 (1:1000; Proteintech, #24072-1-AP), anti-pMLC (1:200 for IF or 1:1000 for WB; CST, #3671), anti-NKp46 (1:200 for IF; eBioscience, #11-3351-82). All the secondary antibodies (1:300 for IF and 1:3000 for WB) were purchased from Life technologies. Phalloidin (1:200) and Hoechst (1:2000) were purchased from Invitrogen. Z-ADD-CMK (Merck Millipore, #368050), LysoTracker® Red DND-99 (Invitrogen, #L7528) and Y27632 (MCE, # HY-10071) were purchased and used according to manufacturer’s instructions.

Cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.5% Na-deoxycholate, 0.1% SDS, 1 mM EDTA, and protease inhibitor cocktail) for 10 min on ice. Lysates were centrifuged at 12,000 rpm for 10 min, and the supernatants were collected. Twenty μg Rhotekin-RBD Protein GST Beads (Cytoskeleton RT02, Denver, CO, USA) or PAK-GST Protein Beads (Cytoskeleton PAK02, Denver, CO, USA) were washed three times using RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.5% Na-deoxycholate, 0.1% SDS, 1 mM EDTA, and protease inhibitor cocktail). Cell lysates (800 μg) were incubated with the beads at 4 °C for 1 h. After incubation, the beads were washed three times using wash buffer (25 mM Tris pH 7.5, 30 mM MgCl₂, and 40 mM NaCl). Two times loading buffer (100 mM Tris-HCl, pH 6.8, 200 mM dithiothreitol (DTT), 4% SDS, 0.2% bromophenol blue, and 20% glycerol) were added to suspend the beads and boiled for 10 min at 95 °C. Active RhoA, active Rac1, or active Cdc42 was then detected by western blot by using anti-RhoA (Cytoskeleton ARH04, Denver, CO, USA), anti-Rac1 (Cytoskeleton ARC03, Denver, CO, USA), or anti-Cdc42 (Cytoskeleton ACD03, Denver, CO, USA).