Alphaimager 2200 system

The level of phosphorylation was determined in cell lysates collected from one million vector control and shMUC2-2.2 cells that were treated with recombinant human IL-6 (25 ng/mL) (R&D Systems, Minneapolis, MN, USA) for 30 min. Relative phosphorylation levels were determined using Human Phospho-RTK Arrays (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Cell lysates were used immediately or stored at −80 °C and were incubated with a human phosphorylation array panel antibody cocktail at RT for 1 h. After blocking, the membranes were incubated with cell lysates at 4 °C overnight on a rocking platform. The membranes were subsequently washed and incubated with a freshly diluted detection antibody cocktail for 2 h at RT on a rocking platform. The membranes were subsequently washed and incubated with diluted streptavidin conjugated to horseradish peroxidase (HRP) for 30 min at RT on a rocking platform. The membranes were developed with Chemi Reagent Mix and exposed to X-ray film. The signal intensity of the array was scanned and quantified by densitometry using an AlphaImager 2200 system (Alpha Innotech) and normalized to the positive control.

The cells were harvested, washed twice with cold phosphate-buffered saline (PBS) and lysed in buffer [50 M Tris-HCl (pH 7.4), 150 M NaCl, 2 M EDTA and 1% NP-40], containing protease inhibitors. The protein concentration was quantified using a bicinchoninic acid protein measurement kit (Shenneng Bocai Biology, Co., Ltd., Shanghai, China). A total of 30 μg extract was subjected to SDS-PAGE and then electrophoretically transferred to a nitrocellulose membrane, which was blocked with 5% (w/v) dried skimmed milk-tris-buffered saline and tween 20 [TBST; 10 M Tris-HCl (pH 8.0), 150 M NaCl and 0.05% Tween 20] for 1 h at room temperature. Subsequently, the membrane was incubated for 2 h with antibodies against RECK (Santa Cruz Biotechnologies, Inc., Santa Cruz, CA, USA; at 1:1000 dilution) and MMP-2 (Santa Cruz Biotechnologies, Inc.; at 1:1000 dilution), respectively, in TBST with 5% skimmed milk. Subsequent to being washed in TBST, horseradish peroxidase-conjugated anti-rabbit IgG (Santa Cruz Biotechnology, Inc.) was used as a secondary antibody (1:10,000, in TBST with 2% bovine serum albumin incubated for 1 h). Each sample was also probed with an anti-GAPDH antibody (Santa Cruz Biotechnology, Inc.) as a loading control. Protein bands were visualized by the Alpha Imager 2200 system (Alpha Innotech, San Leandro, CA, USA).

Total cell lysates were prepared and analyzed by SDS-PAGE as previously described^{43 (link)}. For quantification, the bands were measured using an AlphaImager 2200 system (Alpha Innotech, San Leandro, CA, USA) and were normalized to the band density of β-actin. MUC2 expression was quantified and is presented as the MUC2 to β-actin ratio. These experiments were repeated using three independent batches of cell clones or cell lysates. The quantitative data are presented as the values relative to those in control cells. For more detailed information, please see the Supporting Information.