Anti pias1

Colon tissues (n = 6) were quickly shredded and mixed with protease inhibitor cocktails, which were randomly selected from every group. The mixture was homogenized by ultrasound on crushed ice and centrifuged at 13000g at 4°C for 10 min; the supernatant was extracted; and the protein concentration was determined by BCA protein assay. Equal amounts of total protein (60 μg) were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane using a semidry transfer slot (Bio-Rad, USA). After blocking with 5% nonfat dried milk for 1 h, the membrane was incubated with proportionally diluted antibodies at 4°C overnight. The antibodies included rabbit anti-GAPDH (1:2,000), anti-BCL-2 (1:2,000), anti-BIM-1 (1:1,000), anti-caspase-3 (1:500), anti-BAX (1:1,000), anti-PP2A (1:1,000), anti-β-casein (1:2,500), anti-caveolin-1 (1:1,000), anti-Pim-1 (1:1,000), anti-JAK1 (1:1,000), anti-JAK3 (1:1,000), anti-PIAS1 (1:2,000), anti-PIAS3 (1:2,000), anti-Socs-1 (1:1,000), anti-P-STAT5 (1:1,000), and anti-STAT5 (1:1,000; Abcam). Then, samples were incubated with a corresponding secondary antibody (Abcam) for 1 h. After washing three times with TBST, the blots were visualized by the Proteogel imaging system (FluorChem M, ProteinSimple, USA) and quantified with the Quantity One System (Bio-Rad).

CAM assay was performed as described elsewhere [50 (link)] with slight modifications. For onplant preparation, native, non-pepsinized type I rat tail collagen (BD Bioscience, Bedford, MA) was neutralized with 0.2 M NaOH solution and mixed with 10 × DMEM medium. 5 × 10⁵ PC3 or PC3-DR cells were added to 50 μL of this solution. Collagen-onplants with or without doxycyclin (1 μg/ml) were applied to CAMs and incubated for 5 days. Xenografts were analyzed under a stereomicroscope with a digital camera (Olympus SZX10, Olympus E410, Vienna). For histological analysis, onplants were excised from the CAM, fixed in 4% paraformaldehyde, and processed for paraffin sectioning and IHC. The following antibodies were used: anti-PIAS1 (1:400; Abcam), anti-Mcl1 (1:200; Santa Cruz), and anti-Ki67 (1:100; DAKO, Glostrup, Denmark).