Fig. 7

Analysis of robo3var1 and robo3var2 MO efficacy and specificity. Western Blot analysis using anti-Myc (A, B) and anti-Robo3 (C) antibodies. (A) Coupled in vitro transcription–translation in rabbit reticulocyte lysates, using myc-tagged robo3 constructs as templates, in the absence of the corresponding MO or increasing concentrations of the corresponding MO from 5 nM to 20 μM. Forty-micromolar robo3var2 specific MO was added to robo3var1 reaction mixture (MO swap, top row) and vice versa (MO swap, bottom row). (B) Coupled in vitro transcription–translation reactions for Robo3var1 and Robo3var2 in the absence of the corresponding MO, 20 μM of the corresponding MO and 20 μM of the corresponding 4 mm control MO. (C) Total protein extracted from 11 hpf embryos injected with either a mixture of robo3var1 and robo3var2 MOs (MO) or a mixture of robo3var1 4 mm and robo3var2 4 mm control MOs (4 mm MO). Anti-actin antibody (Actin) was used as a protein loading control.