Maspin

Fetal bovine serum (FBS) and trypsin (0.25% w/v) were purchased from Hyclone (Thermo Fisher Scientific Inc. Rockford, IL). Topotecan (TOPO) was purchased from 21st Century Global E-Commerce Network (East Sussex, UK). Dimethyl sulfoxide (DMSO), sulforhodamine B (SRB), TRIS buffer, acetic acid, ECL western blotting substrate for chemiluminescence were obtained from Bio-Rad (Hercules CA, United States ). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and RNase A were purchased from Sigma-Aldrich Inc (St. Louis, MO). Mouse anti-human antibodies (MMP-9, MMP-1, Ang-2, VEGF, Maspin and c-Fos were purchased from Santa Cruz Biotechnology (uPA; H77A10, MMP-1; SB12e, Ang-2; F-1, VEGF; JH121, Maspin; E10 Santa Cruz, CA) and Cell Signaling Technology (PAI-1; D9C4, uPAR; D7X2N, MMP-9; D6O3H, c-Fos; 9F6, Danvers, MA). Goat anti-Rabbit IgG (H = L) Secondary antibody, HRP, was purchased from Cell Signaling Technology; 345,897 (Danvers, MA). β-actin was purchased from Sigma-Aldrich; A5316. All glass and plasticware were purchased from VWR (Radnor, PA).

Protein extracts were obtained from pieces of tumors or from HaCaT cells. Total protein extracts (30 μg) were subjected to SDS/PAGE. The separated proteins were transferred to nitrocellulose membranes (Amersham, Arlington Heights, IL; BioRad, France) and probed with antibodies against IKKα (NB100-56704 Novus Biologicals); c-Myc (Biolegend, CA, USA); Maspin, Actin, EGFR, P-EGFR (Tyr1176), p65, GAPDH (Santa Cruz Biotechnology, Inc. Europe); α-Tubulin (Sigma-Aldrich, MO, USA); E-Cadherin (BD Bioscience, NJ, USA). p100/p52 (Cell Signaling Technology, USA) and MMP-9 (Merck Millipore, Darmstadt, Germany). In all cases samples were subjected to luminography with the Supersignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Inc., Illinois, USA).