Fig. 2
Dysbindin interacts with p150glued affecting microtubule association and complex integrity. a Co-immunoprecipitation of dysbindin and p150glued. Lysates from HEK293 cells transfected with GFP-hDysbindin and FLAG-p150glued constructs were immunoprecipitated with anti-FLAG antibody. Immunoprecipitates were analyzed by western blotting with anti-GFP and anti-FLAG antibodies. b Co-localization of dysbindin and mitochondria in the axon of DIV 10 primary cortical neurons. Neurons transfected with GFP-mDysbindin and MTS-mCherry were stained with anti-GFP antibody. Overlapped signals are indicated by arrow heads. Scale bars represent 20 μm (left) and 5 μm (right). c Endogenous dysbindin detected from the mitochondrial fraction of DIV 11 primary cultured neurons. Dysbindin was detected by western blotting with anti-dysbindin antibody. Mitofilin and α-tubulin were used as markers for mitochondrial and cytosolic fractions, respectively. d Co-localization of dysbindin and p150glued along the microtubules in the axon of DIV 10 primary cortical neurons. Neurons transfected with mRFP-C1, FLAG-mDysbindin and GFP-p150glued were stained with anti-FLAG (red) and anti-α-tubulin (blue) antibodies. Scale bars represent 20 μm (left) and 5 μm (right). e Microtubule co-sedimentation assay for p150glued and dynein IC in mouse brain tissue. Polymerized tubulin in the pellet fraction (P) and non-polymerized tubulin in the supernatant (S) were subjected to western blotting with anti-p150glued and anti-dynein IC antibodies. f Quantification of the protein level of p150glued and g the protein level of dynein IC in the pellet fraction normalized by that of α-tubulin. h Co-immunoprecipitation of p150glued and dynein IC upon dysbindin co-expression. Lysates from HEK293 cells transfected with indicated constructs were immunoprecipitated with anti-GFP antibody. Immunoprecipitates were analyzed by western blotting with anti-FLAG and anti-GFP antibodies. i Quantification of the protein level of co-immunoprecipitated p150glued normalized by immunoprecipitated dynein IC. j Co-immunoprecipitation of p150glued and dynamitin upon dysbindin co-expression. Lysates from HEK293 cells transfected with indicated constructs were immunoprecipitated with anti-FLAG antibody. Immunoprecipitates were analyzed by western blotting with anti-GFP and anti-FLAG antibodies. k Quantification of the protein level of co-immunoprecipitated p150glued normalized by immunoprecipitated dynamitin. Asterisks indicate the protein of interest. All results are presented as the mean \(\pm\) SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 from Student’s t test