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Fig. 4. Activated MAP-kinase levels in wild-type and sur-6(sv30) mutant backgrounds. Western blots were performed using L4 animals. Membranes were probed with MAPK-YT antibody (Sigma) specific for dually phosphorylated MAP kinase, then stripped and reprobed with K23 antibody (Santa Cruz Biotechnology) which detects total MAP kinase for use as a loading control. Experiments were carried out in triplicate. (A) One representative western blot is shown. The mpk-1/map-kinase gene produces a 45 kDa soma-specific isoform in larvae (M. H. Lee and T. Schedl, personal communication). MAPK-YT reactive bands were absent in mek-2(h294lf) animals (Ohmachi et al., 2002). lin-45(ku112) animals were included as a positive control for reduced MPK-1 ERK phosphorylation. (B) Quantitation of three independent western blots was carried out using a BioRad GS670 imaging densitometer. Bands visualized by the K23 antibody were used as the loading control for the normalization of intensities of the bands visualized by the use of MAPK-YT.





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