Figure 4

DLC1 affects NF-κB activity is dependent on AJs stability. (A) C4-2-B2 cells, transduced with Ad-DLC1 or transfected with deletion mutant DLC1 Δ340-435 (lacking the α-catenin-binding region), were grown in media with or without calcium, and immunostained for E-cadherin and DLC1. Scale bar = 20 μm. (B) C4-2-B2 cells transduced and stably transfected with Ad-DLC1 and sh-α -catenin were grown in media with and without calcium for 24 hours, and stained for phospho NF-κB, DLC1 and α -catenin. Scale bar = 20 μm. (C) Luciferase reporter assay confirms increased NF-κB activity in C4-2-B2 cells grown in calcium-negative media. (D) Cell extracts from C4-2-B2 cells transduced and stably transfected with Ad-DLC1 + GFP (lane 1), Ad-LacZ + GFP-E-cadherin (lane 2) or Ad-DLC1 + GFP-E-Cadherin, were immunoprecipitated with anti-GFP antibodies and western blotted with anti-NFκB-antibodies. Result of the whole cell extract is shown in the right. (E) Membrane and cytosolic fractions of NF-κB in the LacZ and DLC1 transduced C4-2-B2 cells.