2.5. Evaluate the suppression effect of regorafenib on tumor progression proteins by Western blot

About 3 × 10⁶ cells TSGH 8301 cells were incubated in a 100‐mm culture dishes overnight and then treated with 0, 15, or 30 μM regorafenib for 48 h, respectively. Total cells and tumor tissues were collected and gently re‐suspended in lysis buffer [50 mM Tris–HCl (pH 8.0), 120 mM NaCl, 0.5% Nonide P‐40] for sonication and centrifuged as described previously and supernatant was used for measuring total protein by the Pierce BCA protein assay kit (Thermo Fisher Scientific) with bovine serum albumin (BSA) as the standard.16 Total protein was electrophoresed on SDS polyacrylamide gels and then electrotransfered onto PVDF membrane (EMD Millipore), washed and incubated with primary antibodies (anti‐MMP‐9, ‐XIAP, ‐VEGF, ‐CyclinD1, ‐p‐ERK, ‐t‐ERK, ‐p65‐NF‐κB, ‐NF‐κB, ‐p‐p38, ‐t‐p38, and ‐β‐actin). After washed, the membranes were incubated with HRP conjugated anti‐rabbit IgG or anti‐mouse IgG. Immunoreactive proteins were visualized and detected by Immobilon Western Chemiluminescent HRP Substrate (Pierce, Rockford, IL, USA). The band intensities of protein were quantified using the Image J (version 1.50; National Institutes of Health, Bethesda, MD, USA).