Genotypic procedures are described in the supplemental experimental procedures. All animal treatments were done in accordance with the regulations and policies of the Johns Hopkins Animal Care and Use Committee. Antibodies were used at the following dilutions: anti-IIH6 (1:100, Millipore), anti β-dystroglycan (1:100, Santa Cruz), 2H3 http://www.selleckchem.com/products/3-deazaneplanocin-a-dznep.html (1:3,000, concentrated ascites, DSHB),
anti-Nestin (1:100, DSHB) anti-L1 (1:250, Millipore), anti-Laminin (1:1000, Sigma), anti-Perlecan (1:500, Millipore), anti-Collagen IV (1:500, Southern Biotech). In situ hybridization was performed using standard procedures and the following probe for dystroglycan (nucleotodes 562–1,034 of NM_010017). Slit1 and Slit2 probes were described previously ( Yuan et al., 1999). Analysis of dystroglycan glycosylation and laminin binding activity in various tissues by Western blotting was performed as described ( Satz et al., 2010), and are detailed further in the supplemental experimental procedures. Duvelisib mouse To label commissural axons at the floor plate, open book preparations from E13 embryos were unilaterally injected with DiI and imaged 16–24 hr later. The behavior of commissural axons was scored
as normal, stalled, straight, or random (axons projecting both anteriorly and posteriorly following floor plate exit). For quantification of the ventrolateral funiculus area, the area occupied by L1 positive axons in the ventral funiculus and lateral funiculus was calculated using ImageJ using the motor exit point as the demarcation between ventral and lateral funiculi. Three different forelimb levels sections were quantified per animal, and at least three animals
were used for each genotype. To test binding between dystroglycan and Slit, COS7 cells were transfected with constructs encoding α-dystroglcyan-Fc (DG-Fc), AP alone, AP-Slit2 LRR (amino acid postion 273–505 of human Slit2), AP-Slit2 Cterm (amino acid position 1122–1529 of human Slit2), or AP-Slit2 Cterm AVA. Secreted ligands were collected from the supernatant, concentrated, and dialyzed in binding buffer (50 mM first Tris-HCl [pH 7.5], 150 mM NaCl, 2.5 mM MgCl2, 2.5 mM CaCl2). For DG-Fc binding experiments, DG-Fc was incubated overnight with the indicated ligands, then recovered by binding to protein A/G beads for two hours, washed five times in binding buffer, and eluted by boiling in sample buffer. For analysis of Slit binding to endogenous dystroglycan, Slit ligands were incubated overnight with WGA-enriched fractions isolated from mouse brain tissue that had been dialyzed overnight in binding buffer. Ligands contained a C-terminal 6× His tag and were recovered by a two-hour incubation with Ni-NTA beads, washed five times in binding buffer, and eluted by boiling in sample buffer. For COS7 AP-binding, cells expressing either full-length dystroglycan or full-length Robo-1 were incubated with the indicated ligands.
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