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Fig. 3 | BMC Plant Biology

Fig. 3

From: Phosphatase ABI1 and okadaic acid-sensitive phosphoprotein phosphatases inhibit salt stress-activated SnRK2.4 kinase

Fig. 3

ABI1 inhibits kinases from all SnRK2 groups in vitro and in planta. a Recombinant SnRK2.4 (1 μg) was incubated with increasing amounts of PP2Cs for 30 min at 30 °C and kinase activity was analyzed by in-gel kinase assay using MBP as substrate. Dephosphorylation of SnRK2.4 was monitored by immunoblotting using specific anti-P-SnRK2 antibodies. Recombinant kinases SnRK2.8 and SnRK2.6 (as positive control) were incubated with increasing amounts of phosphatases for 30 min at 30 °C and the kinase activity was monitored by in–gel kinase assay with MBP as substrate. SnRK2 phosphorylation status was visualized by immunoblotting with specific anti-P-SnRK2 antibodies recognizing a specific phosphorylated residue in the kinase activation loop (Ser-158 in SnRK2.4, Ser-175 in SnRK2.6, and Thr-158 in SnRK2.8). In parallel, visualization of proteins in samples used for the Western blotting was performed by staining with Coomassie Brilliant Blue, CBB. Data represent one of two independent experiments showing similar results. b EGFP-SnRK2s were co-expressed with c-Myc-ABI1 in Arabidopsis protoplasts isolated from the T87 cell line. The protein level of kinases studied and ABI1 in extracts from protoplasts treated with 300 mM NaCl was monitored by immunoblotting; the activity of the kinases was analyzed by in-gel kinase activity assay using MBP as substrate. c Arabidopsis T87 cells and T87 cells expressing StrepTag-ABI1 were exposed to 500 mM NaCl. Activity of SnRK2.4/SnRK2.10 in the cell extracts was monitored by immunocomplex kinase activity assay, using MBP as substrate. The level of StrepTag-ABI1 and SnRK2.4/SnRK2.10 proteins was monitored by immunoblotting. Autorad, autoradiograph; CBB, Coomassie Brilliant Blue

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