Cytoskeletal alterations in granulocytes of MRP14^-/- mice. (A) Lysates of granulocytes obtained from MRP14^+/+ or MRP14^-/- mice were separated by 15% SDS-PAGE and stained by Coomassie blue (Coo) or processed for [⁴⁵Ca] overlay (⁴⁵Ca²⁺). In all assays MRP14^-/- granulocytes lack MRP14 and present with only a weak band of MRP8. (B) Lysates of granulocytes obtained from MRP14^+/+ or MRP14^-/- mice were separated in microtubule content (right) versus free-tubulin content (left), loaded on an 8% SDS-PAGE, electroblotted on nitrocellulose membrane, and stained with a mAb against α-tubulin. The figure shows representative blots and mean data of 5 independent experiments obtained by densitometric scanning of protein bands. (C) BM-derived granulocytes of MRP14^+/+ (left) or MRP14^-/- mice (right) were cultured for 2 hours on fibronectin-coated LabTec chamber slides. Cells were processed as described in “Materials and methods” and stained with a mAb against α-tubulin. (D) Detergent-soluble proteins from whole-cell lysates of MRP14^+/+ and MRP14^-/- granulocytes were processed for Western blot analysis as described in “Materials and methods,” and nitrocellulose membranes were stained with polyclonal antibodies against Rac1, RhoA, or Cdc42, respectively. Data are presented as described in panel B. Using an mAb against actin we confirmed equal protein loads in the common control by Western blotting (upper panel). The figure shows representative blots and mean data of 5 independent experiments obtained by densitometric scanning of protein bands. Controls of individual experiments were set to 100%. (E) Granulocytes of MRP14^+/+ and MRP14^-/- mice were either left untreated or stimulated with 0.5 mM arsenite for 5 minutes at 37°C. Activation was stopped by addition of ice-cold 2 × lysis buffer and lysates were processed for Rac1, RhoA, and Cdc42 activation assays as described. Equal loading of protein was controlled by a mAb against actin (upper panel). Data are presented as described in panel D.