Fig. 4. FIP distribution in cell fractionation experiments and coimmunoprecipitation of filamin and FIP. (A) 2x10⁸ AX2 cells were developed for 15 hours on phosphate agar, opened by freezing and thawing (Lys), and cytoplasm and membrane fractions were separated by centrifugation at 10,000 g into supernatant (S10) and pellet (P10). The supernatant (S10) was further fractionated by centrifugation at 100,000 g into supernatant (S100) and pellet (P100). The fractions were resolved in 8% polyacrylamide SDS gels and blotted onto nitrocellulose membranes. Blots were incubated with mAb K12-454-2 for FIP detection and filamin-specific mAb 82-454-12. Both proteins are mostly present in the cytosolic fraction and to a lesser extent in the membrane sediment. (B) FIP (a) and filamin (b) can be coimmunoprecipitated from AX2 cell homogenates. In (a), immunoprecipitation was performed with filamin-specific mAb 82-454-12, and the immunoblot containing the immunocomplexes was probed for the presence of FIP with mAb K12-454-2. In (b), immunoprecipitation was performed with FIP-specific mAb K12-454-2, and filamin was detected in the immunoprecipitate using mAb 82-454-12. The immunocomplexes were resolved by SDS-PAGE (10% acrylamide). The bands observed at approximately 170 kDa in (a) are breakdown products of FIP. Likewise, the band below 94 kDa in (b) is a breakdown product of filamin. Both proteins are highly susceptible to proteolysis. The bands at about 60 kDa (star) are the immunoglobulin heavy chains. The homogenates were obtained from cells that were allowed to develop on phosphate agar plates for 12 hours.