Blots were probed at 4��C overnight with primary antibodies in 5% milk/TBST. The antibodies used for Western blotting were CD133, CD29, Musashi-1, ABCG2, TERT and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, Santa Cruz, CA). Two-DE and silver-staining Cultured SW1116 cells were harvested, washed with PBS, and lysed in a lysis selleck Pazopanib buffer containing 8 mol/L urea, 4% CHAPS, 40 mmol/L Tris, 65 mmol/L DTT, 2% Bio-Lytes+ and centrifuged at 25 000 �� g for 1 h at 4��C. Protein concentrations were measured by a modified Bradford assay. All samples were stored at -80��C for electrophoresis. Two-DE was performed using the PROTEAN IEF and PROTEAN xi II systems (Bio-Rad, Hercules, CA, USA). Total protein (80 mg) was run in an IEF system using a 17 cm pH 3 – 10 ReadyStrip (Bio-Rad, Hercules, CA, USA).

The total Vh was 47 000-52 000. Following IEF separation, gel strips were equilibrated with buffer I containing 6 mol/L urea, 30% glycerol, 2% SDS, 1% DTT, followed by buffer II (DTT was replaced with 2.5% IAA), each for 15 min. The equilibrated gel strips were placed on top of a 12% T slab gel and sealed with 0.5% agarose. SDS-PAGE was performed for 30 min at a constant current of 10 mA and then at 25 mA until bromophenol blue reached the bottom of gels. The separated proteins were visualized with silver diamine-staining. For preparative 2-DE, 400 ��g of total proteins was separated as described above. The total Vh of IEF was 90 000-120 000. Proteins were detected with modified silver-staining compatible with MS analysis.

Experiments (from cell culture to 2-DE) were performed in triplicate. Image acquisition and statistical analysis Silver-stained 2-DE gels were scanned at an optical resolution of 84.7 ��m perpixel using a GS-710 imaging densitometer (Bio-Rad, Hercules, CA, USA). Digitized images were analyzed with the PD Quest 7.1 software package (Bio-Rad, Hercules, CA, USA). Following spot detection, a matchset including the three batches of SW1116 cells was built. A reference gel was selected from one of the SW1116 gels, and unmatched protein spots of the member gels were automatically added to the reference gel. The raw quantity of each spot in a member gel was divided by the total quantity of valid spots in the gel. Quantitative analysis of SW1116 gels was performed by Student��s t-test.

Protein identification Protein spots were excised from gels, destained and washed until the gels became clear. The spots were kept in 0.2 mol/L NH4HCO3 for 20 min, dried by lyophilization, and digested overnight Anacetrapib in 12.5 ng/mL trypsin in 0.1 mol/L NH4HCO3. Peptides were extracted three times with 50% ACN (Fisher, Fair Lawn, New Jersey, USA), 0.1% TFA (Merck, Schuchardt, Hohenbrunn, Germany) and dried in vacuum. The peptide mixture was dissolved in 0.1% TFA and desalted using a C18 ZipTip (Millipore, Bedford, MA). The eluted peptides in 0.1% TFA/50% ACN mixed with an equal volume of 0.