Fig. 4. MKP3 regulates its own expression by regulating ERK MAP kinase. (A) Western blot of protein extracted from untreated somites (lane 1), somites electroporated with EGFP vector (lane 2) or electroporated with hMKP3-GFP vector (lane 3); 15 µg was loaded in each lane. Asterisks on the right identify the EGFP protein (lane 2) and the hMKP3-GFP fusion protein (lane 3). -tubulin served as a loading control. (B) PCR performed on plasmid DNA with specific primers. Human MKP3-GFP plasmid was detected with hMKP3-GFP primers but not with cMkp3 primers (lane 1). Chick Mkp3 plasmid was detected with cMkp3 primers but not with hMKP3-GFP primers (lane 2). (C) RT-PCR on cDNA obtained from somites expressing EGFP vector (lane 1) or hMKP3-GFP fusion protein (lane 2). (B,C) Templates used indicated at the top, primers used indicated on the left. (D) Immunohistochemistry on a frontal section using MF20 antibody detecting myosin heavy chain (green) and dpERK anti-body detecting active phosphorylated ERK MAP kinase (red). White arrowheads indicate rostrocaudal boundaries between somites. Dorsal root ganglia indicated by a blue arrow; dorsal sclerotome region indicated by a yellow arrow. (E) Section of somites electroporated with a GFP vector (in red) and hybridised with a probe detecting MyoD (dark blue). Arrows indicate extent of tissue electroporated, including dermomyotome, myotome and dorsal sclerotome. (F-I) Manipulating dpERK levels in somites results in loss of scleraxis transcripts, the following vectors were electroporated: (F,G) hMKP3-GFP, (H,I) hMKP3^KIM-GFP. Single and double in situ hybridisation of electroporated embryos with the probes indicated on each panel. (F) Mkp3, bracket indicates detection of high levels of human transcripts, which crossreact; (G) scleraxis and hMKP3-GFP detected using a GFP probe (n=19/22); (H) scleraxis and hMKP3^KIM-GFP detected using a GFP probe (n=8/8); (I) GFP fluorescence.