Figure 9.

In Vitro Phosphorylation of ALMT4 S382 by MPK6 and MPK4.

Thiophosphorylation assays of GST-tagged native and phosphosite S382A mutant C-terminal ALMT4. GST-ALMT4^WT (WT) and GST- ALMT4^S382A (S382A) were incubated in the presence of Bn-ATPγS as cofactor with GST-MPK4 or GST-MPK6 (see Methods). The GST-MPK recombinant proteins used were sensitive to Bn-ATPγS as a thiophosphate donor (analog-sensitive [AS]) or were wild type, as a negative control. Phosphorylated ALMT4 (phosphoALMT4) was visualized by an anti-thiophosphate ester-specific antibody (α-TPE), and the GST fusion proteins were visualized by an anti-GST antibody (α-GST) to test for equal loading.