Horseradish peroxidase conjugated anti rabbit or anti mouse secondary antibody

Protein lysates were prepared by lysing the HCT116/5-Fu cell lines or tumor tissues in lysis buffer [50 mM Tris–HCl pH 6.8, 5 mM EDTA, 2% sodium dodecyl sulfate (SDS), and 5% glycine]. Protein concentration was estimated by BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts (30 µg) of protein samples were fractionated by SDS-polyacrylamide gel electrophoresis on a 12% polyacrylamide SDS gel at 80 V for 2 hours. Then, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) at 200 mA for 3 hours. The membranes were blocked for 1 hour with 5% skim milk in 0.1% TBST (20 mM Tris pH 7.6, 150 mM NaCl, and 0.05% Tween-20) followed by incubation with primary antibodies at 4°C overnight. The next day, the membranes were washed three times for 15 minutes in TBST, followed by incubation at room temperature with horseradish peroxidase-conjugated antirabbit or antimouse secondary antibody (Santa Cruz Biotechnology Inc., Dallas, CA, USA, dilution 1:5,000). Primary antibodies used rabbit antihuman EIF3G (Bethyl Laboratories, Inc., Montgomery, TX, USA; cat no A301-757A; 1:1,000), mouse antihuman GADPH (Santa Cruz Biotechnology; cat no sc47724; 1:1,000), mouse anti-MRP (Santa Cruz Biotechnology; cat no sc-136447; 1:1,000), and rabbit anti-MDR1 (Abcam, Cambridge, UK; cat no ab129450; 1:1,000).

Whole cell lysates were prepared, and Western blotting was performed as described (17) (link). The antibodies were purchased as follows: neurocan and phosphacan from Chemicon Inc., NG2 from Upstate USA Inc., phospho-ROCK from Bioss, horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody, RhoA, phospho-RhoA, ROCK, xylT1, xylT2, Raf, and phospho-Raf from Santa Cruz Biotech., MLC, phospho-MLC, Akt, phospho-Akt, STAT5 and phospho-STAT5 from Cell Signaling Technology Inc., and β-actin from Sigma Chemical Co..

Following treatment, cells were collected and centrifuged and whole-cell lysates were prepared using a lysis buffer. Total protein concentration was determined using a DC protein assay kit (Bio-Rad, Hercules, CA, USA). Thirty micrograms of protein was directly separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride (PVDF, Bio-Rad Laboratories). Blocked membranes were then incubated with primary antibodies overnight at 4℃. B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X protein (BAX), ERK, phospho-ERK, JNK, and phosphor-JNK purchased from Cell signaling Technology (Beverly, MA, USA); and caspase-3 and caspase-9 from Biovision (Milpitas, CA, USA) were used at a 1:1000 dilution. Subsequent to washing, the membranes were incubated with a horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody (Santa Cruz, Biotechnology, Santa Cruz, CA, USA). The blots were re-probed with anti-β-actin antibody as a loading control. Protein bands were visualized using an enhanced chemiluminescence system (ECL™; Amersham, Little Chalfont, UK) and band intensities were quantified using the Luminescent image analyzer (LAS 4000 mini; Fujifilm, Uppsala, Sweden). All experiments were independently repeated three times.

A375 cells were collected after coculture with either 1–2 μM SAG or 0.5% DMSO culture medium for 48 h. The proteins in the cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride membranes and exposed to the appropriate antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, USA) overnight. The blots were visualized by an enhanced chemiluminescence (ECL) method according to the recommended procedure using horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, USA). All experiments were repeated at least three times independently.

Total protein was extracted from HCC cells using RIPA lysis buffer (Beyotime, Nantong, P.R. China) according to the operating instructions. A total of 30 μg of protein was separated by 10% SDS-PAGE electrophoresis and transferred to nitrocellulose membranes (Amersham, Little Chalfont, UK). After being blocked in Tris-buffered saline containing 5% nonfat milk powder, the membranes were incubated with primary antibodies: anti-CMTM3, anti-E-cadherin, anti-N-cadherin, anti-vimentin, anti-p-JAK2, anti-JAK2, anti-p-STAT3, anti-STAT3, and anti-GAPDH (1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, TX, USA). Subsequently, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody (1:2,000 dilution; Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h at room temperature. Signals were detected on x-ray film using an electrochemiluminescence detection system (Pierce, Rockford, IL, USA).