Fig. 3. Endogenous CENPF and syntaxin 4 colocalize in murine cells. (A) There is significant overlap of endogenous murine CENPF (mCENPF) and syntaxin 4 expression throughout the cytoplasm in C2C12 myoblasts (merge). There is a high degree of colocalization in the perinuclear region, but CENPF has a broader distribution extending further into the cell periphery. (B) 3T3-L1 pre-adipocytes also demonstrate similar expression patterns of CENPF and syntaxin 4 to that seen in C2C12 cells. However, there is higher expression of both endogenous proteins at the cell periphery than in C2C12 myoblasts. (C) 3T3-L1 cells were differentiated as described in Materials and Methods. Both endogenous CENPF and syntaxin 4 appear to have high levels of expression distributed throughout the cells. All images are from confocal microscopy. Bar, 10 µm. (D) Murine CENPF forms an endogenous complex with syntaxin 4. Endogenous protein complexes were analyzed using C2C12 cell lysates for co-immunoprecipitation analysis with Sepharose beads alone, -SNAP-25 antibody, syntaxin 4 antiserum, or IgG antibody alone. After precipitation, elution and western blotting, the blot was probed with -CENPF antibody. Lane 1 demonstrates the presence of CENPF in the lysate. Lane 2 demonstrates the absence of precipitation with beads alone. Lane 3 is a positive control, showing that CENPF precipitates with SNAP-25. Lane 4 shows that CENPF precipitates with syntaxin 4. Lane 5 demonstrates the lack of precipitation with non-immune IgG.