Fig. 1.

Myo9b expression in macrophages and rescue of the Myo9b^−/− phenotype. (A) Myo9b, but not myosin IXa (Myo9a), was detected in resident peritoneal F4/80⁺ cells (F4/80 is a specific marker for mouse macrophages) by Western blot analysis. Neither Myo9b nor Myo9a expression could be detected in Myo9b^−/− macrophages. (B) Live-cell differential interference contrast (DIC) and fluorescence images (100 × 100 μm) of WT and Myo9b^−/− macrophages after overnight incubation. Cells were labeled with Alexa Fluor 488-conjugated anti-F4/80 antibodies. (C) Activity of Rho-family GTPases. GST-pull down and Western blot analysis of RhoA, Rac1, and Cdc42 activities in WT versus Myo9b^−/− macrophages. (D) Schematic diagram showing physiological (Myo9b) and pharmacological (TAT-C3 and Y-27632) inhibition of the Rho signaling pathway. (E) Western blot analysis of phosphorylated cofilin (p-cofilin) and myosin light chain (p-MLC) in macrophages. (F) Time-lapse DIC images (100 × 100 μm) of Myo9b^−/− macrophages after the application of the Rho-specific inhibitor TAT-C3 (50 μg/mL) or ROCK inhibitor Y-27632 (10 μM). (G) Mean 2D cell area of Myo9b^−/− and WT macrophages before and 60 min after applying TAT-C3 or Y-27632. *P < 0.05 (before versus after treatment, paired t test) and data obtained from three to four independent paired experiments per group.