Mertk Deficiency Affects Macrophage Directional Migration via Disruption of Cytoskeletal Organization
Figure 3
Mertk-/- macrophages exhibit unpolarized distribution of filamentous actin and myosin II, and decreased FAK phosphorylation.
Peritoneal macrophages from the WT (A) and Mertk-/- (B) mice were prepared following the procedure described in the Materials and Methods. After fixture in 4% paraformaldehyde for 10 min at RT, cells were blocked in 1x PBS containing 3% normal donkey serum, 0.5% BSA and 0.5% Tween-20, for 1 hr, then incubated with mouse monoclonal antibodies against Myosin II (1:200 dilution, Abcam, MA) and TRITC-conjugated phalloidin (Sigma-Aldrich, MO) in the blocking buffer at +4°C overnight. After washed for 3x with 1xPBS plus 0.5% Tween-20, the cells were incubated in the Alexa Fluor 488-conjugated donkey anti-mouse secondary antibody at RT for 1 hr. Myosin II and F-actin were labeled green and red, respectively. Nuclei were counterstained blue with DAPI (Roche Diagnostics, Germany). Microscopic images were captured and analyzed on a confocal laser-scanning microscope (Leica, Germany). Scale bars in (A) and (B), 5 μm. (C) Western blotting shows decreased FAK phosphorylation at tyrosine 397 after addition of apoptotic T cells for the indicated time points. GAPDH was used as control for indication of equal protein loading.
