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Fig. 3. The N-terminal domains of Nlp and ninein bind the dynein-dynactin complex. (A) Whole cell extracts were prepared as described in the Materials and Methods and incubated in the presence of GST-Nlp full length, GST-Nlp N terminus and C terminus, GST-ninein N terminus or GST-GM130cc. The bound fractions were analysed by blotting with antibodies against dynactin subunits. Dynein was detected using antibodies against the dynein intermediate chain (DIC). The amount of total protein extract loaded was 1/80 of the amount of the bound fractions. The left panel shows a representative Coomassie Blue-stained gel to illustrate GST-fusion proteins. (B) Endogenous Nlp was precipitated from HEK293T cell extracts using rabbit polyclonal anti-Nlp antibodies. Rabbit pre-immune antibodies were used as control. Immunoprecipitates were analysed by blotting using antibodies against the dynactin subunits p150Glued, p50-dynamitin and Arp1.





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