Un scan it gel ver 6

At the end of treatment, the cells were lysed in the ice-cold whole cell extract buffer (pH7.4) containing 250 mM NaCl, 50 mM HEPES, and 0.1% NP-40. The protease inhibitors including 1 μg/ml aprotinin, 0.5 μg/ml leupeptin, and 100 μg/ml 4-(2-aminoethyl) benzenesulfonyl fluoride were added to the cell suspension. The lysate was vibrated for 30 min at 4°C and centrifuged at 12,000 rpm for 10 min. The protein concentrations were determined by the BCA protein assay kit (Pierce, Rockford, IL). Total protein extracts were prepared for western blot analysis using specific SSEA-1 and actin antibodies. Briefly, equal amounts of proteins in samples were subjected to electrophoresis using 10% sodium dodecyl sulfate-polyacrylamide gels. After electrophoretic transfer of proteins onto polyvinylidene fluoride membranes, they were sequentially hybridized with primary antibody and followed with a HRP-conjugated second antibody. Finally, the protein bands were visualized followed by detection with a chemiluminescence kit (PerkinElmer Life and Analytical Sciences, Boston, MA). To verify equal protein loading and transfer, actin was used as the protein loading control. The gel digitizing software, Un-Scan It gel (Ver.6.1, SilkScientific, Inc., Orem, UT), was used to analyze the intensity of bands in X-ray film.

Total cellular RNA was purified by ZR RNA Miniprep (ZYMO research, Irvine, CA) according to the manufacturer's protocol. RNA concentrations were determined by spectrophotometry (Eppendorf, Humburg, Germany). cDNAs were synthesized by SuperScript^TM III oligo reverse transcriptase with oligo-dT 12-18 primer. Each reverse transcript was amplified with actin as an internal control. The following primer pairs were used for amplification: β-III-tubulin: 5′-CTGGAGCGCATCAGCGTATAC-3′ and 5′-ATCTGCTGCGTGAGCTCAGG-3; Actin: 5′-TGTATTCCCCTCCATCGTGG-3′ and 5′-CTCTTTGATGTCACGCACGA TTTC-3′. RT-PCR was performed by a DNA thermal cycler, 5331/Mastercycler gradient (Eppendorf, Hamburg, Germany). The initial denaturation was performed at 95°C for 5 min, followed by 20 cycles at 95°C for 1 min, 56°C, for 1 min, and 72°C for 1 min; and 72°C for 6 min. The PCR products were visualized on 1.5% agarose gels with novel juice staining under UV transillumination, and photograph was taken by a camera (DH27-S3, Medclub, Taoyuan, Taiwan). To verify equal cDNA loading and transfer, actin was used as the protein loading control. The gel digitizing software, Un-Scan-It gel (Ver.6.1, SilkScientific, Inc., Orem, UT), was used to analyze the intensity of bands.