escribed. MFG. EGFP. IRES. puro and the retroviral vector MFG. I��B. IRES. puro, which encodes a supersuppressive mutant form of I��BM, were generated and infected into gastric cancer cells, as described previ ously. Pooled puromycin resistant cells were used for further analysis. STAT3 siRNA transfection STAT3 siRNA and scrambled siRNA were pur chased from Santa Cruz Biotechnology. STAT3 siRNA or control siRNA was then transfected into gastric cancer cells using LipofectAMINE Plus according to the manufacturers instructions. Preparation of nuclear and cytoplasmic extracts Cells were scraped and lysed in cold lysis buffer A, incubated on ice for 10 min, centrifuged, and the cytoplasmic extracts obtained were transferred to fresh tubes.
For nuclear extracts, the pelleted nuclei were washed in 1 mL buffer A without NP 40 and resuspended in 50 uL cold lysis buffer B. They were then extracted on ice for 10 min with occasional vortexing. The lysate was centrifuged at 170 g at 48 C for 2 min, and the supernatant was collected as nuclear extracts. Immunoblotting Cell lysates were prepared in 100 200 uL of 1x sodium dodecyl sulfate lysis buffer. Protein contents were measured using BCA Protein Assay Reagent. Entinostat Equal amounts of proteins were loaded onto a 10% discon tinuous SDS polyacrylamide gel and electrophoretically transferred to PVDF membranes blocked with 5% non fat dry milk in phosphate buffered saline Tween 20 for 1 h. The membranes were then incubated at 4 C overnight with or without 2 h incubation at room temperature with one of the following primary antibodies, anti RelA, anti phospho Ser536 RelA, anti STAT3, anti phospho Tyr705 STAT3, anti E cadherin, anti Snail, anti MMP9, anti B actin and anti TFIIB.
Horserad ish peroxidase conjugated anti rabbit IgG or anti mouse IgG was used as a secondary anti body. Enhanced chemiluminescence was used to detect the immunoreactive proteins. Equal protein loading was confirmed by B actin or TFIIB. Transient transfection and luciferase reporter assay The NF ��B luciferase reporter plasmid contains a 5x NF ��B response element fused to luciferase. The STAT luciferase reporter plasmid contains four copies of the sequence GGTTCCCGT AAATGCATCA fused to luciferase. SNU 638 cells were transiently co transfected with 0. 4 ug of luciferase reporter plasmid and 0. 4 ug of B galactosidase vector, an internal control, using LipofectAMINE Plus.
Twenty four hours after transfection, assays for luciferase and B galactosidase were carried out using a Dual Luciferase Reporter Assay System. Luciferase activity was measured on an AutoLumat LB 9505c luminometer and was normalized by B galactosidase activity. Luciferase ac tivity in control cells was arbitrarily set to 1. Immunofluorescence staining Cells were cultured on chamber slides. After 24 h, cells were fixed with 4% paraformaldehyde, permeabilized with 0. 5% Triton X 100 for 5 min, and blocked with 5% normal donkey serum. After blocking, cells were incubated over night at 4