Morphology and RhoA activation in fibrinogen-adhered Mgks. (A) Mgks were isolated from the bone marrow cells of the femur and/or tibia of mice using a bovine serum albumin gradient after 5 days of culture with recombinant murine TPO. GP expression was determined via flow cytometry. The data are presented as the mean and SD (n = 3 each). ∗∗P < .01 and ∗P < .05 (1-way ANOVA). (B) Cultured murine Mgks (3.0 × 10⁴) were added to fibrinogen-coated coverslips and incubated at 37°C for 20 minutes. The cells were then fixed, permeabilized, and stained with TRITC-conjugated phalloidin (red), Alexa Fluor 488–labeled CD41 (green), and DAPI (4',6-diamidino-2-phenylindole; blue), followed by observation under a confocal microscope (Olympus FV3000) (40×). Scale bars represent 50 μm. (C) Cultured murine Mgks were serum starved for 24 hours and plated on fibrinogen-coated dishes. After 15 minutes of incubation, the cells were solubilized with lysis buffers provided by the manufacturer. The amount of activated RhoA was measured using an ELISA-based assay kit, G-LISA, that quantifies the amount of the active GTP-bound form of RhoA. Measurements were taken for 3 mice of each phenotype in 2 independent experiments (left). The total RhoA expression in murine Mgks was measured via immunoblotting (right). Data are representative of 2 independent experiments. MFI, mean fluorescence intensity.