CIP4 localizes with WASP and the actin cytoskeleton network in MKs and platelets. (A) CIP4 is present in megakaryocytic cells. Western blotting of human or murine platelets, murine megakaryocytes, or human megakaryocyte cell line CHRF-288-11 demonstrated the presence of CIP4 at the expected 70 to 80 kD size. A weaker 100 kD band is of unknown nature. (B) CIP4-GFP localizes throughout the cytoplasm and at the plasma membrane. Human CIP4 tagged with GFP was expressed in CHRF-288-11 cells treated with PMA using the Amaxa Nucleofector II. Images were taken with a Leica DM 4000B microscope and analyzed by LAS (Leica Application Suite) software (Leica, Wetzlar, Germany). (Objective 100×/numerical aperture 1.3, scale bar = 10 um). (C) CIP4 and WASP colocalize in cultured WT megakaryocytes. Confocal microscopy with using a Nikon Eclipse C1Si confocal microscope (objective 40×/numerical aperture 1) and EZ-C1 software (Nikon, Tokyo, Japan) showed the distribution of CIP4 (Alexa-488) or WASP (Alexa-594). When merged, colocalization occurred in both cytoplasm and proplatelets. Upper image: anti-CIP4 staining (secondary antibody conjugated with Alexa-488) Middle image: anti-WASP staining (secondary antibody conjugated with Alexa-594). Lower image: merge and 4′,6 diamidino-2-phenylindole. (objective 40×/numerical aperture 1, scale bar = 10 um). (D) CIP4 localization to actin cytoskeletal network in platelets. Platelets were activated with thrombin and lysed in 1% Triton X-100. The Triton insoluble fraction was resuspended in RIPA detergent buffer, centrifuged, and the actin cytoskeleton was collected. The Triton X-100 soluble fraction was centrifuged, and the pellet was resuspended in RIPA, centrifuged, and membrane cytoskeleton was collected from the supernatant. Western blot was performed with antibodies directed against either WASP or CIP4, which are found in the cytosol in platelets and translocates from the membrane cytoskeleton to the actin cytoskeleton. WCL, whole cell lysates.