Figure 3
Analysis of the specificity of JFA1 and JFA2 compounds to ABA receptors based on ABA-dependent inhibition of ABI1 activity. (a) Principle of ABI1-dependent dephosphorylation assay of SnRK1.1 by the AlphaScreen using anti-phospho AMPK antibody. (b) ABI1-dependent dephosphorylation analysis of biotinylated SnRK1.1. The amount of FLAG-ABI1 was set with the volume ratios (μL) to biotinylated SnRK1.1 as 0–2:10. (c) Analysis of ABA receptor mediated inhibition of ABI1 activity. Biotinylated SnRK1.1 was incubated with or without C-terminal AGIA-tagged ABA receptors (PYR1, PYL1, 2, 4, 5, 6, 8, 9, 10, 11, and 12), FLAG-ABI1, or 1 μM ABA. Then, the phosphorylation level of SnRK1.1 was analyzed by using the AlphaScreen. The relative SnRK1.1 phosphorylation signal was expressed as a relative value with the signal of the mock control (SnRK1.1 only) as one. (d) Analysis of PYR1 mediated inhibition of ABI1 activity by immunoblot. Biotinylated SnRK1.1 was incubated with (+) or without (−) PYR1-AGIA, FLAG-ABI1, or 1 μM ABA. Phosphorylation of SnRK1.1 was analyzed by using the anti-phospho AMPK antibody. Asterisk indicates non-specific band. (e) Analysis of ABA receptor mediated inhibition of ABI1 activity in the presence of ABA, JFA1 or JFA2. Biotinylated SnRK1.1 was incubated with C-terminal AGIA-tagged ABA receptors (PYR1, PYL1, 2, 4, 5, 6, 8, 9, 10, and 11) and FLAG-ABI1. PYR1, PYL1, PYL4, and PYL6 were incubated with various concentrations of each compound (1, 10, or 100 μM), and PYL5, PYL8, PYL9, PYL10, and PYL11 were incubated with various concentrations of each compound (0.01, 0.1, 1, 10, or 100 μM). Relative ABI1 activity was expressed as a relative value with the signal of the DMSO (1%) control as one. Error bars represent standard deviations (n = 3).
