Fig. 4

Detection of c-SRC total protein and phosphorylated forms by isoelectric focusing. Plots (d–f) show values after normalization to HSP70 levels. Symbols in plots: Red; KRAS mutated, green; BRAF mutated, blue; wild type (WT) with regard to KRAS and BRAF, black; unclear for KRAS and BRAF. a. Immunoblotting of selected tissue samples with antibodies against c-SRC pY418, c-SRC pY527 and total SRC protein. For upper panel, loading control β2M was same as Fig. 2a. GAPDH was used as a loading control for lower panel. Control; HUVEC cell lysate was used as a positive control. b. Representative electropherogram showing c-SRC total protein and phosphoprotein peaks. Phosphorylated peaks (blue line) were identified by virtue of their sensitivity to lambda phosphatase digestion (green line). Inset; electropherogram showing HSP70 run in parallel. c. Representative electropherogram showing c-SRC pY527 peaks. d. Plot of combined c-SRC peak (P1–P6) areas in normal mucosa, stage II and stage IV CRC. e. Plot of phosphorylated c-SRC peak (P1–P5) areas. f. Plot of the ratio pSRC/SRC