Western blot analysis Protein was collected from cultured HepG2, SMMC7721, HepG2/ADM and SMMC7721/ADM selleck chemicals Pazopanib cells and its concentration was measured (protein assay dye, Bio-Rad). Then, the protein was denatured in a LDS sample buffer for 5 min at 95��C, run on SDS-PAGE (NUPAGE, 4%-12% Bis-Tris, Invitrogen, Carlsbad, CA, USA) and blotted onto PVDF membranes (0.2 ��m, Invitrogen). Membranes were blocked with 5% dry milk in TBS-T (TBS containing 0.05% Tween 20) for 1 h at room temperature and incubated overnight at 4��C with antibodies against ERK1, ERK2, or phospho-ERK1/2 (Thr202/Tyr204) (Cell Signaling Technology, Inc., Danvers, MA, USA).

After incubation with the respective primary antibodies, the membranes were exposed to species-specific horseradish peroxidase-labeled secondary antibodies at room temperature, and developed using the ECL plus Western blotting reagent (GE Healthcare, Little Chalfont, UK) and Fuji Film LAS-1000 equipment (Fuji Film, Tokyo, Japan). Parallel membranes were incubated with 1:5000 rabbit monoclonal antibodies to GAPDH (Cell Signaling Technology, Inc.) and HRP-coupled rabbit anti-mouse secondary antibody. Primary and secondary antibody solutions were prepared in a PBS solution containing 2% bovine serum albumin and 0.1% Tween-20. After incubation with antibodies, the membranes were washed 3 times for 5 min in PBS containing 0.1% Tween-20. Calculation and statistics were performed using the ImageJ 1.37 software. Statistical analysis Statistical analysis was performed using Student��s t-test to compare the two groups and ANOVA was used with Dunnett��s post-test for multiple comparisons when the three groups or more were compared.

P < 0.05 was considered statistically significant. The results were expressed as mean �� SE. Values were analyzed Brefeldin_A using the statistical package SPSS for Windows Ver.11.5 (SPSS Inc., Chicago, IL, USA). RESULTS Determination of MDR Each step of developing MDR HepG2/ADM and SMMC7721/ADM cells took 7-8 wk. MDR was maintained by culturing the cells with 0.2mg/L ADM. Cytotoxicity assay found that HepG2/ADM and SMMC7721/ADM were resistant not only to ADM but also to multiple anticancer drugs. Among them, fluorouracil (5-FU), cyclophosphamide (CTX), cisplatin (CDDP), mitomycin (MMC), and vincristine (VCR) were tested in our study. Their lethal dose (IC50) was significantly higher for HepG2/ADM and SMMC7721/ADM cells than for non-resistant parental cells (Figure (Figure11 and Table Table11). Table 1 Determination of IC50 and resistance index of different anticancer drugs (mean �� SD) Figure 1 Measurement of cellular sensitivity to anticancer drugs and parental cells.