Fig. 5. Western blot analysis of signaling pathway intermediates activated in
Mpl-expressing clones after long-term TPO stimulation. Cells were washed three
times and stimulated with 10 ng/ml of TPO for 3 or 9 hours. Cells were lysed
and 60 µg of protein per lane was fractioned by SDS-PAGE. Lysate of clone A
deprived of cytokines for 3 hours was used as a control. (A) Activation of
STAT-5, ERK and AKT after 9 hours of TPO stimulation was analyzed as described
in Fig. 1. (B) Expression level
of Bcl-XL and BAD phosphorylation analyzed by immunoblotting with
anti-Bcl-x and anti-BAD antibodies. As a control, blots labeled with the
anti-Bcl-x antibody were stripped and relabeled with a mouse anti-actin
antibody. ns, no stimulation. Positions of unphosphorylated (BAD), singly
(BADP1) and doubly phosphorylated BAD (BADP2) are indicated.