Fig. 2
From: Loss of MBNL1-mediated retrograde BDNF signaling in the myotonic dystrophy brain
MBNL1 interacts with cytoplasmic DYNLL2 in an RNA-independent manner. (a) Examination of FLAG-MBNL1ΔEx5 and GFP-DYNLL2 binding by immunoprecipitation using an anti-GFP antibody in Neuro2A cells treated with or without RNase treatment. (b) Immunoprecipitation analysis using an anti-GFP antibody to determine binding between endogenous ubiquitinated MBNL1, TrkB and dynein intermediate chain (DIC) in cells expressing GFP-DYNLL2. SE, shorter exposure time. LE, longer exposure time. (c) Identification of the DYNLL2 binding domain on MBNL1. Truncated MBNL1 constructs are illustrated on top. An anti-GFP antibody was used to perform immunoprecipitation. ZnF, zinc-finger domains. (d) Examination of the distribution of cytoplasmic MBNL1 and DYNLL2 (top) or TrkB (bottom) in neurons using plasmids expressing EGFP-tagged or mCherry-tagged MBNL1ΔEx5 with DYNLL2-mCherry or EGFP-TrkB. Arrowheads indicate colocalization region along the neurite. (e) Distribution of MBNL1, TrkB and DYNLL2 proteins in the homogenate (H), nuclei and other large debris (P1), crude synaptosomal fraction (P2), cytosolic soluble fraction (S3), light membrane fraction (P3), synaptosomal membrane fraction (LP1), soluble fraction (LS2), and crude synaptic vesicle fraction (LP2). PSD95 and synaptophysin were used as markers for synaptic compartments. Scale bar: d, 20 μm