Immobiline drystrip

2D-PAGE and mass spectroscopic identification of differentially expressed spots was undertaken essentially as previously described [30 (link)]. Briefly, A549 cells treated with vehicle, andrographolide or ZAD-1 for 24 h were harvested and proteins prepared, and a total of 250 µg were loaded onto Immobiline Drystrips (pH 3–10, NL, 7 cm) for isoelectric focusing using Ettan IPGphore II system (Amersham Biosciences Amersham, UK). The second dimension electrophoresis was undertaken on 12.5% polyacrylamide gels. Gels were stained with 0.1% colloidal Coomassie Brilliant Blue G 250 with 20% methanol for 24 h and destained with milli-Q water for 48 h. The resultant gels were scanned under visible light at 300 µm/pixel resolution. Image data were analyzed using Image Master™ 2D Platinum software version 7.0 (GenBio, Geneva, Switzerland). Each spot was evaluated on intensity, volume and area. Statistical analysis was performed by one-way analysis of variance (ANOVA), with a p-value of 0.05 being considered significant. The experiment was performed as three independent biological replicates for each condition. After tryptic in-gel digestion, peptide masses were determined by LC-MS/MS using SYNAPT high definition MS (HDMS) (Waters Corp., Milford, MA, USA).

The chemical reagents and experimental procedure for 2D gel electrophoresis were described in our previous study [22 (link)]. After treatment, the total proteins of the cells were extracted and precipitated by 10% trichloroacetate in acetone. Their concentrations were measured by the Bradford assay with bovine serum albumin as the standard sample for normalization. Prior to the 2D-PAGE analysis, protein samples were suspended in a rehydration solution and then applied to Iso-Electric Focusing (IEF) in the pH 3–10 immobilized-gradient strips (Immobiline Dry Strips, Amersham Biosciences, Uppsala, Sweden) with an Ettan IPGphor II apparatus (Amersham Biosciences). Using 10% SDS-PAGE gels, it was possible to carry out the two-dimension electrophoresis.

HMVEC-L cells were harvested by centrifugation and then disrupted with lysis buffer containing 5 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1% Triton X-100, and 2 mM PMSF. The cell lysate was centrifuged at 12,000 Χ g for 30 min, and the supernatant fraction was collected. Protein concentrations were determined using a BCA assay kit (Pierce). Immobiline DryStrips (Amersham Biosciences) were used for isoelectric focusing, which was carried out with 1 mg of the extracted protein on an IPGphor system (Amersham Biosciences). After IEF separation, the proteins were separated in the second dimension by SDS-PAGE. For image analysis, the gels were visualized with Coomassie Brilliant Blue G-250 according to the manufacturer’s instructions. The 2-D gels were scanned with an ImageScanner (Amersham Biosciences) in transmission mode. Spot detection and matching were performed using ImageMaster 2D version 5.0 (Amersham Biosciences). Digitized images were analyzed using the ImageMaster program to calculate the 2-D spot intensity by integrating the optical density over the spot area (the spot “volume”) and normalized. The values were normalized and then exported to SPSS 18.0 for statistical analysis.

Intracellular proteins were extracted and separated by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D SDS-PAGE; 2DE) as described previously [64 (link)]. Isoelectric focusing was carried out on 18 cm Immobiline Dry Strips pH 4–7 (Amersham Pharmacia Biotech, Sweden) followed by gel electrophoresis on 14% polyacrylamide gels (185 × 200 × 1.0 mm). Gels for Coomassie staining were loaded with 150 μg protein. Gels for fluorescent staining were loaded with 50 μg protein, and proteins were pre-labelled with the T-Rex Labelling kit (NH DyeAGNOSTICS) before rehydration (see “Staining procedures” below). The gels were then fixed in the appropriate fix solution as recommended by the respective stain manufacturer (Coomassie stain 2% acetic acid and 40% ethanol and fluorescent stain 10% acetic acid and 50% ethanol). Gels for Coomassie staining were fixed for at least 1 h and gels for fluorescent staining were fixed overnight, protected from light. All gels were produced in triplicates from separate cell cultures.

The chemicals and reagents used for 2D gel electrophoresis were as described in our previous study 20. Erinacine A–treated and untreated HCT‐116 cells were harvested and protein extraction was carried out. Prior to 2D‐PAGE analysis, the protein concentration was measured using the Bradford assay with bovine serum albumin as the standard sample for normalization; following cell lysis, the total cell protein was precipitated by 10% trichloroacetate in acetone. Protein samples were suspended in rehydration solution and subjected to isoelectric focusing in 13‐cm, nonlinear, pH 3–10 immobilized‐gradient strips (Immobiline DryStrips; Amersham Biosciences, Uppsala, Sweden) in an Ettan IPGphor II apparatus (Amersham Biosciences). The second dimension electrophoresis was carried out using 10% SDS‐PAGE gels.