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. 2019 Aug 13;31(11):2682–2696. doi: 10.1105/tpc.19.00058

Figure 7.

Figure 7.

The Kinase Activity of BIN2 Is Regulated by Cold Stress.

(A) and (B) Mutation of BIN2 and its homologs attenuates ICE1 degradation during cold stress. Total proteins from ICE1-OE18 transgenic plants or bin2-3 bil1 bil2/ICE1-OE5 (grown on half strength MS for 2 weeks) treated at 4°C for the indicated period were extracted and subjected to immunoblot analysis. ICE1 was detected with anti-HA antibody. Actin was used as the internal control. The IOD values of ICE1 and Actin were quantified using the software Image-Pro Plus (Media Cybernetics) to calculate the relative protein abundance of ICE1 (IODICE1/IODActin) in relation to IODICE1/IODActin at 0 h. Representative picture is shown in (A), and quantitative analysis is shown in (B). Data are the means ± sd of three independent experiments. *P < 0.05 (Student’s t test).

(C) and (D) ICE1S94A is more stable than wild-type ICE1 during cold stress. The experiment was performed using ICE1-OE/ice1-2 and ICE194A-OE/ice1-2 plants. The relative protein abundance of ICE1 was calculated as described in (A). Representative blot (C) and quantitative analysis (D). Data are the means ± sd of three independent experiments. *P < 0.05 (Student’s t test).

(E) and (F) In-gel kinase assays of BIN2. Total proteins were extracted from Super:BIN2-MYC transgenic plants treated at 4°C for the indicated period and incubated with anti-MYC agarose. A portion of the immunoprecipitated protein sample was used to perform an in-gel kinase assay using the common substrate MBP as a substrate (top), and a portion was used for the immunoblot assay (bottom). BIN2 kinase activity was detected by autoradiography, and BIN2 protein levels were detected with anti-MYC antibody. The IOD values of BIN2 kinase in the autoradiograph and protein abundance were quantified using the software Image-Pro Plus (Media Cybernetics), and the relative kinase activity was calculated as IODAuto/IODMYC. The relative kinase activity was set in relation to IODAuto/IODMYC at 0 h. Representative blot (E) and quantitative analysis (F). Data are the means ± sd of three independent experiments. *, nonspecific bands. WT, wild type.