Journal: Journal of Molecular Signaling
Article Title: Glycogen Synthase Kinase-3beta regulates Snail and beta-catenin during gastrin-induced migration of gastric cancer cells
Figure Lengend Snippet: Effect of GSK3β inhibition on G17-induced Snail expression and β-catenin nuclear translocation . (A) AGSE cells were treated with (+) or without (-) 100 nM G17, following an overnight pretreatment with either none (lanes 1, 2), 5 μM (lanes 3, 4) or 10 μM (lanes 5, 6) AR-A014418. Western Blot analysis was then performed with the antibodies indicated. (B) Luciferase (with Snail-luc) and β-Gal assays were performed as in 2D following a 1 hour pretreatment with AR-A014418. (C) AGSE cells were co-transfected with Snail-luc and β-Gal vectors along with either Empty vector (lanes 1, 2), GSK3β-S9A mutant vector (lanes 3, 4) or GSK3β-K/A mutant vector (lanes 5, 6). Luciferase and β-Gal assays were performed after G17 treatment as in 2D. Each transfection (3B, 3C) was performed in triplicate, and the data represent the mean ± SD of at least two independent experiments. (D) Upper Panel : Confluent AGSE cells were treated with G17 for 8 hours after an overnight pretreatment with none (lanes 1, 2), or AR-A014418 (lanes 3, 4) or SP600125 (lanes 5, 6). At the end of treatment, nuclear protein was isolated and subjected to Western Blot analysis with antibodies against β-catenin, GAPDH (cytoplasmic protein) or Lamin A/C (nuclear protein). Lower Panel : Cells were pretreated as in the upper panel, followed by 1 hour G17 treatment and Western Blot analysis.
Article Snippet: The antibodies utilized were obtained from the following sources: GSK3β, pGSK3βSer9 , AKT, pAKTSer473 , pc-Jun, c-Jun, Snail and Lamin A/C (Cell Signaling Technology, Danvers, MA), GAPDH (Ambion, Austin, TX), β-catenin (BD Biosciences, San Jose, CA), HA.11 (Covance, Berkeley, CA).
Techniques: Inhibition, Expressing, Translocation Assay, Western Blot, Luciferase, Transfection, Plasmid Preparation, Mutagenesis, Isolation