Research Article

Generation of Constitutive Active ERK Mutants as Tools for Cancer Research in Zebrafish

Figure 2

Western blot detection of ERK1/2, P90RSK, and CREB phosphorylation in zf4 cells transfected with PCS2+ERK1 and PCS2+ERK2 (mutated) constructs. (a) Western blotting was performed with total ERK and dpERK. The Ectopic ERKs derived from synthetic mRNAs have a greater MW than the endogenous ERKs, due to the introduction of a small linker, and can therefore be distinguished by size. The bands with higher molecular mass were also detected with anti-ERK 1/2 antibody in cells transfected with mutants but not with the anti-phospho-ERK 1/2 antibody. This confirms that the bands with higher molecular weights are from the transfected ectopic ERK. (b) Western blot detection of phospho-p90Rsk with two different antibodies one targeted against Thr573 and another against Ser380. (c) Western blot detection with the phospho-Creb Ser133 antibody which cross-reacts with phospho-ATF. (d) Band intensities were measured for quantification of western blots ((a)–(c)) dp-ERK(ectopic) p-P90RSK and p-CREB band intensities were corrected for total ectopic ERK synthesis. Mutants were normalized against ERK1-WT or ERK2-WT. WT: protein samples from zf4 cells transfected with wtERK 1 or wtERK 2, *: single mutants , **: double mutants , ***: triple mutants , TEY: , and 0: untransfected zf4 cells, EGFP: zf4 cells transfected with pcs2+EGFP.