Xenbase Image ID: 172384

Figure 2. ERK3 MOs block the translation of ERK3A and/or ERK3B mRNA(s). A, The target sequences of three antisense MOs for Xenopus laevis ERK3 homeologs are shown in rectangles. ERK3 MO1 and MO2 were designed to target ERK3A alone and ERK3B alone, respectively. ERK3 MO3 was designed to target both ERK3A and ERK3B. B-E, The indicated sets of MOs (80 ng), ERK3 mRNAs (1.6 ng) and GFP mRNA (800 pg, control) were injected into the animal regions of all blastomeres at the 4-cell stage. C-terminally Myc-tagged ERK3A (ERK3A-Myc) and ERK3B (ERK3B-Myc) mRNAs comprise the 5’UTR and the coding region to contain the entire MO target sequences. N-terminally Myc-tagged ERK3A (Myc-ERK3A) and ERK3B (Myc-ERK3B) mRNAs contain additional AUG and Myc-encoding sequences upstream of the coding region to avoid MO-mediated translational inhibition. To analyze the ERK3A-Myc and ERK3B-Myc proteins, which were unstable due to proteasome- dependent degradation, animal caps (N=25, each sample) were dissected at stage 9, cultured with 10 μM MG132 (a proteasome inhibitor) until stage 12, and then lysed with 50 μl of lysis buffer. To analyse the Myc-ERK3A and Myc-ERK3B proteins, injected whole embryos (N=10, each sample) were harvested at stage 12 and lysed with 200 μl of lysis buffer. The protein levels were examined by immunoblotting. The data are representative of two or three independent experiments. B, ERK3 MO1 blocked the translation of ERK3A-Myc mRNA but not that of ERK3B-Myc mRNA. C, ERK3 MO2 blocked the translation of ERK3B-Myc mRNA but not that of ERK3A-Myc mRNA. D, ERK3 MO3 blocked the translation of both ERK3A-Myc and ERK3B-Myc mRNAs. E, Myc-ERK3A and Myc- ERK3B mRNAs were MO-resistant. Image published in: Takahashi C et al. (2018) Copyright © 2018. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View

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