Figure 4.
CAP interacts with FLNc. (A) GST-CAP1-405 was used to affinity purify associating proteins. Cell lysates were subjected to pull down by GST or GST-CAP1-405, followed by SDS-PAGE, and stained with Coomassie Blue. Bands were cut out and analyzed by mass spectrometry. The arrow indicates the presence of FLNc specifically in GST-CAP1-405 pull down (lane 4). (B) FLNc is a yeast two-hybrid hit of CAP1-405. (C) HA-FLNc coimmunoprecipitated with myc-CAP when overexpressed in COS-1 cells. WCL, whole cell lysates. (D) Coimmunoprecipitation of endogenous FLNc with CAP in C2C12 cells. (E) CAP interacts with FLNc through the second Ig repeat (FLNc-R2). COS-1 cells were transfected with myc-CAP. Cell lysates were subjected to pull down experiments by GST-fusion fragments of filamins. (F) GST alone or GST-CAP1-405 were used to pull down in vitro translated HA-FLNc or the ΔR2 mutant. The precipitates were subjected to SDS-PAGE and Western blot analysis. Ponceau S staining shows the amount of GST fusion proteins used.