Fig. 5. MIIA interacts with CXCR4 and -arrestin. (A) Direct interaction between CXCR4 and MIIA in protein-protein binding assays. [³⁵S]Met-MIIA pulldown assays were attempted with GST (lanes 1 and 3) or GST-CXCR4 (lanes 2 and 4) and sepharose-bound proteins were resolved by SDS-PAGE. After Coomassie-Blue staining – lane 3 (GST) and lane 4 (GST-CXCR4) – gels were developed by autoradiography (lanes 1 and 2). (B) Direct interaction between -arrestin and MIIA. [³⁵S]Met-MIIA was mixed with recombinant -arrestin 1 and -arrestin 2, precipitated with an anti-pan--arrestin pAb (384-397) and the sepharose-bound proteins were analyzed by autoradiography. (C) Interaction of endogenous MIIA with -arrestin. J77 cells were treated with 300 nM SDF-1 for the indicated periods of time, lysed and precipitated with anti-CD45 (D3/9 mAb) and polyclonal anti-pan -arrestin (172-268). Gels were then analyzed by western blot, revealing both MIIA and -arrestin 1 (mAb 38-44); Lys, lysate. The lane labeled Ab shows unspecific bands from the immunoprecipitating antibody in the absence of cellular lysates. Numbers indicate the quantification of the MIIA band corrected with the amount of -arrestin immunoprecipitated in each lane relative to no SDF treatment. Arrows indicate major myosin bands. Additional upper and lower minor bands correspond to a fraction of the protein retained at the interface with the stacking gel and to a degradation product, respectively.