Figure 5. AnkB interaction with the... | Rockefeller University Press

Figure 5.

$Figure 5. AnkB interaction with the dynactin subunit p62 through DD1320 residues recruits dynein–dynactin to organelle membranes. (A and B) Immunoprecipitation from adult mouse brain homogenates show 220-kD AnkB association with DIC and the dynactin subunits p50 and p62. (C) Coimmunoprecipitation experiments from COS7 cells expressing p62-HA and WT or DD1320AA GFP-AnkB proteins. (D) The amounts of membrane-associated AnkB, β2-spectrin, dynein, dynactin, and kinesin were evaluated by immunoblotting after subcellular fractionation of brain homogenates on a sucrose step gradient. Rab5, LAMP1, and synaptophysin are membrane controls. GAPDH is a loading control. (E) Time-lapse images and kymographs of p62-GFP dynamics in axons of control or AnkB-null neurons. Data also show results of rescue experiments in which WT or mutant DD1320AA AnkB-mCherry was coexpressed with p62-GFP in axons. (F) Immunoprecipitation from COS7 cells expressing GFP–β2-spectrin and AnkB-HA proteins. IP, immunoprecipitation; IB, immunoblotting.$

AnkB interaction with the dynactin subunit p62 through DD1320 residues recruits dynein–dynactin to organelle membranes. (A and B) Immunoprecipitation from adult mouse brain homogenates show 220-kD AnkB association with DIC and the dynactin subunits p50 and p62. (C) Coimmunoprecipitation experiments from COS7 cells expressing p62-HA and WT or DD1320AA GFP-AnkB proteins. (D) The amounts of membrane-associated AnkB, β2-spectrin, dynein, dynactin, and kinesin were evaluated by immunoblotting after subcellular fractionation of brain homogenates on a sucrose step gradient. Rab5, LAMP1, and synaptophysin are membrane controls. GAPDH is a loading control. (E) Time-lapse images and kymographs of p62-GFP dynamics in axons of control or AnkB-null neurons. Data also show results of rescue experiments in which WT or mutant DD1320AA AnkB-mCherry was coexpressed with p62-GFP in axons. (F) Immunoprecipitation from COS7 cells expressing GFP–β2-spectrin and AnkB-HA proteins. IP, immunoprecipitation; IB, immunoblotting.