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Fig. 8. ERK2 is essential for the effect of PLD1 on the formation of lipid droplets, by promoting the phosphorylation of dynein, which increase the association of this protein with lipid droplets. Dynein is essential for the formation of the lipid droplets. (A) NIH 3T3 cells were transfected with PLD1 or GFP as control. After 1 day, the cells were incubated for 2 hours with Ste-Mek113 (25 µM) and stained with Oil Red O. The total area of Oil Red O-stained lipid droplets was determined using BioPix software. The results are given as mean ± s.d. of all cells in 20 randomly selected micrographs (*NS vs control; 
P<0.001 vs control and P=0.001 vs PLD1 + Ste-Mek113; one-way ANOVA). (B) NIH-3T3 cells were incubated for 24 hours with 360 µM oleic acid, harvested and homogenized. The homogenate was incubated with active ERK2 (0.1 µM for 1 hour at 37°C; ERK2) or buffer (Control) and fractionated with the PhosphoProtein purification kit (`phospho-protein affinity column'). The retained fraction (phosphorylated proteins) was collected and analyzed for the indicated proteins by immunoblotting (anti-dynein and anti-caveolin). (C) NIH 3T3 cells were incubated with oleic acid overnight, homogenized by nitrogen cavitation and subjected to gradient ultracentrifugation. The top fraction containing the lipid droplets was isolated and fractionated with the PhosphoProtein purification kit (`phospho-protein affinity column'). The retained (phosphorylated) proteins and unretained (unphosphorylated) proteins were blotted against antibodies to the dynein intermediate chain. (D) NIH 3T3 cells were transfected with ADRP-HAT and incubated with oleic acid (see B). After 24 hours, the cells were homogenized and the homogenate was incubated with active ERK2 (see B) or buffer. ADRP-HAT was precipitated with talon-Dynabeads (ADRP-HAT precipitate) and analyzed by immunoblot against antibodies to dynein. (E) NIH 3T3 cells were microinjected with a monoclonal antibody directed against dynein (17 cells) or a control immunoglobulin (26 cells) and the effect on the total area of Oil Red O-stained lipid droplets/cell determined. (mean ± s.e.m.; P<0.001, Mann-Whitney rank sum test). (F) NIH 3T3 cells were microinjected with a monoclonal antibody directed against dynein (Anti-dynein: 1 µg/ml) or a control immunoglobulin (Control Ig) and stained with Bodipy. The cells were imaged by confocal microscopy at intervals of 30 seconds over a 5-minute period. BioPix software was used to identify fusions as described previously (Bostrom et al., 2005). Results are mean ± s.e.m. of control immunoglobulins (13 different movies) and anti-dynein (16 different movies) (P<0.001, Mann-Whitney rank sum test).
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