Figure 4

Mass spectrometry-based confirmation of secreted laminin-γ2. (A) Preparative gel electrophoresis of conditioned media from SW620 cells (Smad4-deficient) treated with TNFα. An 8% SDS-PAGE was performed with preparative amounts of protein (32 μg) in the middle lane and analytical amounts of protein (16 μg) in the left and right lanes under non-reducing conditions. The left and right lanes were used for Western blotting with a laminin-γ2-specific antibody. Four small slices (1-2 mm) corresponding to the Western blot signals were cut from the preparative gel lane and proteins analysed by mass spectrometry. (B) Amino acid sequence of laminin-γ2. The peptides identified by mass spectrometry in gel bands 1-4 are indicated.