A Resealed-Cell System for Analyzing Pathogenic Intracellular Events: Perturbation of Endocytic Pathways under Diabetic Conditions
Figure 5
Intracellular PI3P was decreased in a p38 MAPK-dependent manner in Db cells.
A. HeLa cells were pretreated with or without 2 µM SB203580 or 2 µM SB202190 at 37°C for 60 min. Semi-intact HeLa cells were incubated with 3 mg/ml WT or Db liver cytosol, an ATP regenerating system, GTP, and glucose in the presence or absence of 2 µM SB203580 or SB202190 at 32°C for 30 min, and then for a further 15 min at 32°C after the addition of 1 µg of GST-2xFYVE recombinant protein. The cells were fixed and GST-2xFYVE was visualized with Alexa488-conjugated antibodies against GST. Bar = 10 µm. B. The fluorescence intensity of GST-2xFYVE was measured as described in Materials and Methods, and the means and standard deviations for the fluorescence intensity are shown in the graph. We performed three independent experiments and counted 100 cells in each experiment. Data were analyzed using one-way ANOVA and Dunnett’s post hoc test, and the P value was < 0.01 (**). C. Measurement of PI3P content in WT and Db cells by lipid blot. D. Western blotting of p38 MAPK and phosphorylated p38 MAPK in liver lysates from three WT or three Db mice. E. The intensities of the bands shown in C were measured and the relative proportion of phosphorylated p38 is shown as a percentage. Means and standard deviations for the relative proportion are shown in the graph. F. Western blotting of p38 MAPK and phosphorylated p38 MAPK in WT and Db cells.