Synergistic induction of laminin-332 in human adenoma cells in response to inflammatory cytokines TGFβ and TNFα. (A) Western blot analysis of heterotrimeric laminin-332 expressed by LT97 colorectal adenoma cells. Proteins (8 μg/lane) prepared from serum-free conditioned media from LT97 cells treated with recombinant TGFβ and TNFα for 48 h as indicated were separated on 3-8% tris-acetate gradient gels (Invitrogen) under non-reducing conditions. The blot was probed with a laminin-γ2-specific antibody (polyclonal antibody 2140, PS-A) and reprobed with a transferrin-specific antibody used as a loading control. The bars indicate the relative signal strength normalized for transferrin. Note that the Odyssey detection system (LI-COR) allows for a direct digital quantification of signals. Similar results were obtained in > three experiments. The same signals, although with less sensitivity, were obtained using a commercial antibody (MAB-19562, Chemicon). (B) Northern blot analyses of the LAMA3, LAMB3 and LAMC2 genes prepared with RNAs from LT97 cells treated with cytokines for 24 h. Quantification of mRNA levels was done by phosphorimage analysis and signal strengths normalized with GAPDH. Similar results were obtained in three experiments.