Figure 3. MK induced cascade in B cells.

B cells derived from C57BL/6 mice were incubated in the presence or absence of MK (100 ng/ml) for various periods. Immediately after stimulation, cells were washed and fast frozen in liquid N₂. The cells were lysed as described in Materials and Methods (A) Lysates were separated on 10% (w/v) SDS-PAGE, and proteins were blotted with anti-p-Akt or anti-Tubulin antibodies. The results presented are representative of at least four different experiments. (B) An aliquot from the lysates was reserved for total Syk analysis. Phosphorylated proteins from the remaining lysate were immunoprecipitated (IP) with an anti-Tyr(P) antibody. Immunoprecipitates and total lysate proteins were separated on 10% (w/v) SDS-PAGE and blotted with an anti-Syk antibody. The results presented are representative of at least three different experiments. (C) RNA was purified from control B cells after stimulation with PBS or MK (100ng/ml) for 6h. Quantitative Real time PCR was performed using primers for Bcl-2 and β-actin. β-actin levels were used to normalize samples for calculation of the relative expression levels of Bcl-2. Results are expressed as a fold change in MK expression in stimulated cells compared to non-stimulated cells, which was defined as 1. Results shown represent an average of at least eight separate experiments. (D) B220+ B cells derived from C57BL/6 mice were incubated in the presence or absence of MK (100ng/ml) for 6h. Cells were lysed, and levels of Bcl-2 and Tubulin were analyzed by western blot analysis. The results presented are representative of at least four different experiments.