Fig. 5.

Defective Cdc42, Rac1 and RhoA activation in Atgl−/− macrophages. a, b, d Wt and Atgl−/− macrophages and (c) MEFs were serum-starved for 3 h and then incubated in DMEM containing 9 ng/ml LPA. Cell lysates were incubated with glutathione-sepharose beads complexed with GST-PBD fusion proteins to pull down GTP-bound Rac1, Cdc42 and RhoA. Precipitates were resolved by SDS-PAGE in parallel with whole cell lysates (total Cdc42/Rac1/RhoA). Protein expression of (a, c) Cdc42 and GTP-Cdc42, (b, c) Rac1 and GTP-Rac1, (d) RhoA and GTP-RhoA was determined by Western blot analysis using specific antibodies. Band intensities were quantified using Image J software, and ratios of two Western blots of activated to total protein expression were determined. Fold changes were calculated relative to the ratios in unstimulated cells. e Wt and Atgl−/− macrophages were plated on fibronectin-coated coverslips and serum-starved for 2 h. Cells were incubated in the absence (control) and presence of SDF-1 (60 ng/ml) and LPA (9 ng/ml) for 10 min at 37°C. Thereafter, cells were fixed and stained with anti-RhoA and AlexaFluor-586 phalloidin to visualize F-actin. Pictures were recorded using an Olympus FSX100 fluorescence microscope. Scale bars 5 μm