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. 2015 May 7;9:15–25. doi: 10.4137/JEN.S22969

Figure 2.

Figure 2

Strumpellin interacts with spartin. (A) Immunoprecipitation of the whole rat brain extracts proteins with spartin antibodies and analyzed by immunoblotting with strumpellin antibodies. (B) Immunoprecipitation with strumpellin antibodies and immunoblotted with spartin antibodies. First lanes show the studied proteins from cell lysates used for immunoprecipitation experiments, second lanes show antibodies used as the “bait” (spartin panel A, strumpellin panel B) incubated with protein A/G, and third lanes show control experiments with protein A/G alone. The 137-kD band represents a full-length strumpellin, and the 80-kD band with an additional faint 68-kD band corresponds to spartin. Very faint bands were also present in lanes Ip-IgG in panels A-1 and B, representing a nonspecific background; however, lanes IP anti-strumpellin and IP anti-spartin with co-immunoprecipitation had more than 100-fold higher activity in both instances. (C) Immunoprecipitation of WASH1 protein with strumpellin antibodies (second lane) and not by spartin antibodies (third lane). A faint nonspecific band was detected in control experiments without addition of specific antibodies (panel C, fourth lane). Panels D1-3 and E1-3 show interactions of WT and studied strumpellin mutations with a full-length spartin and spartin fragments 1–208 and 208–666. All three forms of expressed strumpellin:GFP, WT (lanes 1), N471D mutation (M1, lanes 2) and V629F (M2, lanes 3) interacted with a full length of spartin:myc (panel D1 spartin bait, panel E1 strumpellin bait). Coexpression of strumpellin:GFP and spartin:myc 1–208 did not pull down strumpellin:GFP (panel D2 spartin bait, panel E2 strumpellin bait) but the presence of spartin:myc 208–666 detected all three forms of strumpellin:GFP (panel D3 spartin bait, panel E3 strumpellin bait).