To quantify the densities of the bands, the gray values were measured with the Bio-Rad imaging system. After the values of lamin A/C were normalized by the corresponding values of β-actin, the ratio of the tumour to the
non-tumour gastric tissues was calculated. For real-time MK-2206 manufacturer RT-PCR, each reaction was done on a MX3000P real-time PCR instrument with the SYBR PremixEx Taq™ (Takara, Dalian, China) in a 25 μl reaction system with 1 μg cDNA following the manufacturer’s protocol. All reactions were repeated three times. β-actin was used as an internal control, and measurements between samples were compared by the threshold cycle of amplification (CT). The fold change in expression levels was determined by a comparative CT method using the formula:ΔΔCT(ΔΔCT = (CT find more (lamin A/C) – CT (β-action))learn more cancer – (CT (lamin A/C) – CT (β-action))normal). Primer sequences used for lamin A/C are: forward 5′-CGGTTCCCACCAAAGTTCA-3′ and reverse 5′-CTCATCCTCGTCGTCCTCAA-3′; for β-actin: forward 5′-CACCCAGCACAATGAAGAT-3′ and reverse 5′-CAAATAAAGCCATGCCAAT-3′. The primers were designed between different exons and encompassing large introns to avoid any amplification
of genomic DNA. QPCR was performed for pre-denaturing at 95 °C for 10 seconds, followed by 40 cycles (95°C for 5 seconds and 57.5°C for 20 seconds). Western-blot analysis Western blot was performed on 34 tumour specimens and corresponding adjacent Oxalosuccinic acid non-cancerous samples. The frozen tissues were lysed in RIPA buffer plus protease inhibitors PMSF (Sangon, Shanghai, China), and the resulting insoluble material removed by centrifugation at 12,000 g 4°C for 30 min. After concentration measured by the BCA method, protein samples were electrophoresed on 12% sodium dodecyl sulphate (SDS)-polyacrylamide gels and subsequently transferred to a PVDF membrane (Millipore, Billerica, MA) by electroblotting. After blocking for 1 h in Tris buffered saline (pH 7.6, containing 0.1% Tween and 5% non-fat milk) at room temperature, membranes
were incubated overnight at 4°C with primary polyclonal antibody against lamin A/C (Cell Signaling, Danvers, MA, at 1:1000 dilution), and β-actin (Abcam, Cambridge, UK, at 1:2000 dilution) with gentle shaking. After washing, the membrane was then probed with the appropriate secondary antibody for 60 min at room temperature. Protein binding on the membrane was detected by the enhanced chemiluminescence (ECL) detection system (Pierce, Rockford, IL) according to the manufacturer’s instructions. Then band intensity was measured by densitometry using the Quantity One software (Bio-Rad, Hercules, CA). The protein levels were normalized with respect to β-actin protein level. Immunohistochemistry analysis Sections (4 μm thick) of formalin fixed, paraffin wax blocks were cut onto polylysine-coated microscope slides.