Figure 2
From: Drosophila Dullard functions as a Mad phosphatase to terminate BMP signaling
Dullard decreases BMP-dependent and independent Mad linker phosphorylations.
In all western blots shown Drosophila S2R+ cells were co-transfected with Flag-Mad, +/− Dullard, or +/− Dullard dsRNA and +/− activated-Tkv. (a) Dullard overexpression resulted in a decrease in Mad linker phosphorylation levels at serines 212, 208 and 204 (statistical significance when comparing lanes 1 and 2, pMadS212 P < 0.001 and pMadS204/08 P = 0.004; comparing lanes 3 and 4, pMadS212 P < 0.001 and pMadS204/08 P < 0.001, using the t-test, n = 3 independent blots measured for each). (b) Dullard knockdown resulted in increased levels of Mad linker phosphorylations (statistical significance when comparing lanes 1 and 2, pMadS212 P = 0.014 and pMadS204/08 P < 0.001; comparing lanes 3 and 4, pMadS212 P = 0.014 and pMadS204/08 P < 0.001; using the t-test, n = 3 independent blots measured). (c) C-terminally mutated Mad proteins (Mad-AVA) are linker phosphorylated. These blots demonstrated that Mad-AVA can be linker phosphorylated in the absence of C-terminal activation by the BMP pathway. (d) Phosphatase inactive Dullard mutants (Dul-D66E and Dul-D68E) failed to dephosphorylate the Mad linker domain compared to wild type Dullard.
