Figure 2

Comparison of SW480 and SW620 cells for overall pY levels, SFK activity, Lck expression and activity. Both cell lines are derived from the same patient. SW480 was established from the primary tumor, SW620 from a lymph node metastasis. A – D: 20 μg of TCL from each cell line were separated by SDS-PAGE and western blots (WB) conducted with antibodies as indicated. Arrowheads in A indicate pY bands increased or only detectable in SW620 cells. The asterisk in C indicates a barely visible band that comigrates with the expected size of Lck. Actin levels were analysed as loading control in D. To determine the phosphorylation of Lck in the activation loop (results shown in E), which critically regulates Lck activity, 1 mg of TCL from each cell line was immunoprecipitated with anti-Lck mAb and probed with anti-pY419Src that cross-reacts with several SFK family members, including Lck, due to high conservation of this epitope (further details in [5]). An IgG control of the same isotype was also included to precipitate non-specific binding proteins from 1 mg of TCL of SW620 cells. Note that some of the molecular weight markers used do not run exactly accordingly to the indicated molecular weights of the marker proteins, presumably due to the coupling of dye molecules to them.