Anti mmp7

Anti-TM4SF4 antibody for western blot analysis was purchased from Sigma-Aldrich. Antibodies against phospho-Ser473 AKT, AKT, phospho-ERK, ERK, phospho-NF-kappaB (p65), NF-kappaB, phospho-PI3K, PI3K, PTEN, phospho-Tyr1068 EGFR, EGFR, phospho-IGF1R beta (Tyr1131)/insulin receptor beta (Tyr1146), IGF-1R beta (111A9), and anti-beta-actin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-MMP-2, anti-MMP-7, and anti-MMP-9 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Western blot analysis was performed as described previously [37 (link)]. Antibodies against N-cadherin and E-cadherin were purchased from BD Biosciences.

Total protein extraction from tumor tissues using RIPA buffer (Cell Signaling Technology, Inc. Danvers, MA) and western blotting were performed as described previously studies [18 (link), 19 (link)]. The following primary antibodies were used: anti-Wnt-1, anti-β-catenin, anti-c-myc, anti-cyclin D1, anti-MMP-7, anti-axin-2, anti-APC, anti-c-jun, or anti-β-actin antibodies (Santa Cruz Biotechnology, Inc.). The secondary antibodies were corresponding to the type of primary antibody using anti-mouse or anti-rabbit IgG horseradish peroxidase (HRP)-conjugate (1:1,000 dilution, Cell Signaling Technology, Inc.). Protein bands were visualized by the Enhanced Chemiluminescence kit. The quantification of protein bands were measured by FluorChem Imaging Systems (Alpha Innotech). The data were standardized by β-actin.

Western blotting was performed as described previously 18 (link). Protein samples were collected, concentrated, and SDS-PAGE protein loading buffer was added. Samples were analyzed using SDS-PAGE and then transferred to a PVDF membrane. The membrane was immediately placed in the prepared western washing solution and rinsed for 1-2 min. The membrane was then treated with blocking solution, followed by incubation with the first antibody overnight. The next day it was incubated with the second antibody (1:2500) at room temperature for 2 h. The primary antibodies used were: anti-ATF4 (sc-22800, Santa Cruz, USA, 1:200), anti-β-catenin (sc-393501, anti-Santa Cruz, USA, 1:200), anti-cyclin D1 (sc-20044, Santa Cruz, 1:200), anti-MMP7 (sc-515703, Santa Cruz, 1:200), anti-Histone H3(sc-517576, Santa Cruz, 1:500) and anti-GADPH (sc-59540, Santa Cruz, USA, 1:1000). The membrane was treated with enhanced chemiluminescence (ECL) for protein bands, and the signals were detected using a BioImaging System. To determine the relative protein expression in nucleus and cytoplasm separately, nuclear and cytoplasmic protein samples were prepared by using the Nuclear plasma separation Kit (Beyotime, China).