Figure 6

The MAP kinase p38 indirectly regulates the activation status of Lyn. Shown in panel A are phosphorylation levels of Lyn in presence of a panel of inhibitors with Lyn molecule as loading control. The images in panel B confirm the existence of a co-localized pool of p38 and SHP-1 in unstimulated CH1 cells. Further, increase in the extent of co-localization was also observed after anti-IgM stimulation for 5 min. Panel C shows the Pearson's co-localization coefficient for p38 and SHP1 obtained from the images in panel B. Values obtained in both unstimulated and anti-IgM-stimulated (5 min) are plotted. Experiments were performed in triplicates and mean value ±S.D. is plotted (* indicates p-value < 0.05). Panel D shows the results of an experiment where the BCR was immunoprecipitated from either unstimulated, or anti-IgM-stimulated (5 min) cells. Immunoprecipitates were then resolved by gel electrophoresis and then probed for the presence of either p38, SHP-1 or Lyn by Western blot analysis. Results shown confirm that all of these three molecules were associated with the BCR in both unstimulated and stimulated cells. In addition, these results further confirmed the findings of p38-SHP-1 co-localization in panel B and C. The negative control shown represents a Western blot for Lyn in samples where mouse IgM conjugated to agarose was used for the immunoprecipitation. A similar negative result was also obtained when these samples were probed for presence of either p38, or SHP-1 (not shown). Panel E shows phosphatase activity present in immunoprecipitates of the BCR that were obtained from unstimulated cells either in presence or absence (Normal) of the p38 inhibitor SB203580 (Materials and Methods). The assay was performed in triplicates and the mean value (± S.D.) is given, * indicate statistically significant (p-value < 0.05) increase in phosphatase activity in presence of SB203580.