Pro hgf

Recombinant human HAI-1, recombinant human HGFA, and recombinant human HGF propeptide (pro-HGF) were purchased from R&D Systems. SRI 31215 (10 μM) or Recombinant human HAI-1 (20 nM) were mixed with activated recombinant human HGFA pro-peptide (1nM) in TNC buffer (pH 8.0) and incubated at room temperature for 30 minutes. Recombinant human pro-HGF (40 ng) was added and incubated at 37°C for 1 hour. The reaction was stopped by SDS-PAGE gel sample buffer and samples were boiled and separated by 12% PAGE. Proteins were transferred onto nitrocellulose membrane, blocked with 5% milk and immunoblotted using antibodies that recognize pro-HGF as well as α and β chains of activated HGF.

A431 cells were cultured in 12‐well tissue culture plates. After colonies formed (4‐8 days), cells were serum‐starved overnight and were then treated with pro‐HGF (20 ng/mL; R&D Systems) in the presence or absence of 3‐Cl‐AHPC (0.5 μM). Images of migrating cells were captured at 48 hours after the treatment for 48 hours.

Primary antibodies used were anti-MET, anti-pMET, anti-EGFR, anti-pEGFR, anti-GAB1, anti-pGAB1, anti-AKT, anti-pAKT, anti-pERK1/2, (Cell Signaling Technology) and anti-β-actin (Sigma). The antibody arrays used were Proteome Profiler Human Phospho-Receptor Tyrosine Kinase (RTK) (ARY001B) and Phospho-Kinase Array (ARY003B) kits from R&D Systems. Recombinant HGF and pro-HGF were purchased from R&D Systems. The MET kinase inhibitor, JNJ38877605 was purchased from Selleck Chemicals (Selleckchem). HGF-specific and nonspecific (NSP) siRNAs were purchased from Dharmacon.