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Fig. S3 Translational blocking by gpr161-MO morpholino detected by in vitro cell lysate assay. (A) Two translational blocking morpholinos, targeted against the 5′UTR upstream of the ATG start codon of zebrafish gpr161 open-reading frame, which was fused to V5-6xHistidine epitope. (B,C) Dose dependent inhibition of Gpr161 translation by two independent gpr161 morpholinos (gpr161-MO#36 and gpr161-MO#24) using in vitro cell lysate. No inhibition by 5-base mismatch control morpholino (mismatch-MO#28). Anti-V5 antibody was used to detect the in vitro translation (B) of Gpr161-V5 fusion protein and band intensity (C) was quantitated as % translation compared to control in the graph. No morpholino as control (100%), 10 μM MO#24 (7%), 1 μM MO#24 (56%), 0.1 μM MO#24 (84%), 10 μM MO#28 (79%), 1 μM MO#28 (92%), 0.1 μM MO#28 (79%), 10 μM MO#36 (0%), 1 μM MO#36 (13%) and 0.1 μM MO#36 (40%). (D) Both gpr161 morpholinos (MO#24 and MO#36) gave similar cardiac looping defect. All scale bars were 100 μm.
Reprinted from Developmental Biology, 323(1), Leung, T., Humbert, J.E., Stauffer, A.M., Giger, K.E., Chen, H., Tsai, H.J., Wang, C., Mirshahi, T., and Robishaw, J.D., The orphan G protein-coupled receptor 161 is required for left-right patterning, 31-40, Copyright (2008) with permission from Elsevier. Full text @ Dev. Biol.