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. 2015 Oct 6;56(12):2351–2367. doi: 10.1093/pcp/pcv146

Fig. 9.

Fig. 9

Proteasome-dependent degradation of MKKK18 is ABA-dependent and regulated by ABI1. (A) qPCR analysis of MKKK18 transcript accumulation in response to treatment with 50 µM ABA for 90 min in WT Col-0, MKKK18oe, abi1td and abi1-2 strains. MKKK18 expression levels were determined using three biological replicates and were normalized against 18S rDNA. The results are displayed as mean log2 fold change ± SE (n = 9) of three independent experiments with consistent results. (B) MKKK18 stability in the cell-free degradation assay. GST–MKKK18 was incubated with 100 µg of protein extract from either WT Col-0 or abi1td protoplasts incubated with or without 100 µM MG132 for 6 h in the dark. GST–MKKK18 protein levels at the indicated time points were determined by immunoblotting using anti-GST antibodies. Ponceau S staining confirmed equal loading. (C and D) Half-life plot for cell-free degradation of MKKK18 in WT Col-0 (C) and abi1td (D) extracts. Immunoblot images from each experiment were recorded simultaneously using a G:BOX Chemi XR5 fluorescence and chemiluminescence imaging system (Syngene), and the results were quantified using ImageJ software. (E) CHX treatment suppresses accumulation of MKKK18. Arabidopsis protoplasts expressing 35S:MKKK18-GFP were treated with 3 mM CHX, 3 mM CHX and 100 µM MG132, or given a mock treatment. The blot is representative of four experiments. MKKK18–GFP protein levels were determined by immunoblotting using anti-GFP antibodies. Ponceau S staining confirmed equal loading. MKKK18 protein bands were quantified using ImageJ software and normalized to the control (mock) band (set as 1). (F) MKKK18 protein levels are modified by ABA. GST–MKKK18 was incubated with 100 µg of total protein extract isolated from WT Col-0 incubated with or without 50 µM ABA for 3 h in the dark. GST–MKKK18 protein levels at the indicated time points were determined by immunoblotting using GST antibodies. Ponceau S staining confirmed equal loading. MKKK18 protein bands were quantified using ImageJ software and normalized to the control band (set as 1).