Fig. 7.

ABI1 inhibits MKKK18 activity. (A) Phosphatase activity of recombinant ABI1 and ABI2 proteins. The enzyme reactions were performed in a 50 µl final volume containing 3–5 µg pf GST–ABI1 or GST–ABI2. The results presented are the means from three independent biological replicates. (B) ABI1 inactivates MKKK18. The MKKK18–GFP immunocomplex was incubated with 3 µg of GST–ABI1 and GST–ABI2 (as a negative control), after which the kinase activity was determined with MBP as a substrate. Equal loading was confirmed by Coomassie Brilliant Blue (CBB) staining of MBP. The ³²P-labeled MBP bands were quantified and then normalized against the intensity of the corresponding control band using ImageJ software. Data are means ± SD of the relative band intensities from three independent experiments. An asterisk (*) indicates statistically significant changes determined using Student’s t-test. (C) Recombinant MKKK18 has no autophosphorylation activity in vitro. GST–MKKK18 was incubated with MBP or/and MKK3, without or in the presence of [³²P]ATP. Bands of GST–MKKK18, MKK3–GST and GST–SnRK2.6 are indicated by a filled circle, a triangle and an arrow, respectively. Protein loading was confirmed by CBB staining. The blots shown are representative of three independent trials.