Fig. 6.

Increased ROS production and Rac2 activation in Atgl−/− macrophages. a Wt and Atgl−/− macrophages were incubated in DMEM containing LPA (9 ng/ml) for 0, 2, 5 and 10 min. Cell lysates were incubated with glutathione-sepharose beads complexed with GST-PBD fusion protein to pull down GTP-Rac2. Precipitates were resolved by SDS-PAGE in parallel with whole cell lysates (total Rac2). Band intensities of two Western blots were quantified using Image J software, and ratios of activated to total protein expression were determined. b Wt and Atgl−/− macrophages were plated on fibronectin-coated coverslips for 10 min, fixed and stained with anti-Rac2 antibody and phalloidin AlexaFluor-586. Pictures were taken on an Olympus FSX100 fluorescence microscope. Scale bars 5 μm. c Wt and Atgl−/− macrophages were loaded with 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (DCFDA, 50 μM) for 10 min. Fluorescence was measured in a FlexStation device (excitation: 488 nm; emission: 535 nm). Data are presented as means (n = 3) ±SEM measured in quadruplicate repeats. ***p ≤ 0.001