Rabbit anti human oct 4

The bulk Panc-1 cells, the upper chamber cells, and the lower chamber cells lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Merck Millipore, USA). Membranes were blocked with 5% (w/v) bovine serum albumin (BSA) in TBST for 1 h at room temperature and incubated overnight with primary antibodies at 4°C. They were subsequently incubated with horseradish peroxidase-conjugated second antibodies. The immunoreactive bands were detected by chemiluminescence (ECL Plus, Merck Millipore) and relevant blots were quantified by densitometry using LANE-1D software. For immune detection, the primary antibody preparations used were as follows: rabbit-anti-human-CD24, rabbit-anti-human-ESA, and rabbit-anti-human- Bmi-1 were obtained from Santa Cruz Biotechnology (SantaCruz, CA, USA). Rabbit-anti-human-Oct-4, rabbit-anti-human-E-ca, rabbit-anti-human-Vimentin, rabbit-anti-human-N-ca, rabbit-anti-human-SHH, and rabbit-anti-human-β-catenin were obtained from Cell Signaling Technology (Boston, USA). Anti-β-actin was obtained from Abcam Company (Cambridge, Britain). The secondary antibody preparations either anti-rabbit or anti-mouse were purchased from Boster Biotechnology Company (Wuhan, China).

Lysis buffer (95°C) was used to lyse cells and protein was denatured by boiling at 95°C for 10 minutes before sonication (S-4000 with cuphorn; Misonix) for 2 minutes (80%). Cells were then centrifuged at 10,000×g for 5 minutes at 20°C and supernatant was collected. Lysates were subjected to standard SDS-PAGE followed by blotting onto nitrocellulose membrane (Biorad). Blots were then blocked with 5% milk powder in Tris-buffered saline with Tween (0.1%) and probed with rabbit anti-human OCT4 (Cell Signaling; 1∶1000), anti-GFP (MBL; 1∶1000) or mouse anti-GAPDH (Sigma; 1∶125,000) overnight at 4°C in blocking buffer. The next day membranes were probed with horseradish peroxidase-conjugated secondary antibodies at 1∶10,000 (Jackson ImmunoResearch) for 1 hour and visualized with ECL Prime (GE Biosciences).