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. 2016 Nov 7;173(1):524–535. doi: 10.1104/pp.16.01546

Figure 8.

Figure 8.

Effects of MG-132 on GLK1 accumulation in vivo. A, Effects of MG-132 on the accumulation of GLK1 in wild-type and gun1-101 plants treated with NF. Plants were treated with DMSO (lanes 1 and 4) for 4 d or with NF for 6 d. The NF-treated plants were further treated with additional DMSO (lanes 2 and 5) or 50 μM MG-132 (lanes 3 and 6) for 18 h. Extracted proteins were then resolved by SDS-PAGE, and proteins were probed with antibodies against GLK1 or actin. Note that the exposure time to capture GLK1 signals in wild-type lanes was longer than that in gun1-101 lanes. B, Effects of MG-132 on the accumulation of GLK1 in ppi2-2 mutant overexpressing GLK1 (GLK1ox ppi2-2). Note that only the plants carrying homozygous ppi2 genotype (ppi2 −/−) exhibit damaged plastids and albino phenotype. The GLK1ox ppi2-2 mutant was grown on plates for 7 d. Then, plants were transferred to liquid MS medium. The heterozygous GLK1ox ppi2-2(+/−) plants (green phenotype) were grown in liquid MS medium for 6 d under continuous light and then subjected to DMSO treatment for 18 h. The homozygous GLK1ox ppi2-2(−/−) plants (albino phenotype) were grown in liquid MS medium for 10 d under continuous light and then treated with either DMSO (lane 2) or 50 μM MG-132 (lane 3) for 18 h. Extracted proteins were then resolved by SDS-PAGE, and proteins were probed with antibodies against GLK1 or actin. C, Quantification of GLK1 mRNA and GLK1 protein levels in NF-treated plants (NF) and both NF and MG-132-treated plants (NF + MG-132) shown in (A). GLK1 protein levels were quantified using image acquisition software and normalized to actin levels. The mRNA levels were analyzed by real-time PCR, and the expression levels were normalized to that of ACTIN2. The expression levels in wild-type (left) or gun1-101 (right) plants treated with both NF and MG-132 was set to 1. Error bars shown in mRNA data represent 1 SE of the mean (n = 3). *, P < 0.05 as determined by Student’s t-test. D, Quantification of GLK1 mRNA and GLK1 protein levels in the GLK1ox ppi2-2(−/−) mutant treated with DMSO or MG-132 shown in (B). GLK1 protein levels were quantified using image acquisition software and normalized to actin levels. The mRNA levels were analyzed by real-time PCR, and the expression levels were normalized to that of ACTIN2. The expression level in GLK1ox ppi2-2(−/−) plants treated with MG-132 was set to 1. Error bars shown in mRNA data represent 1 se of the mean (n = 3). ns, not significant. WT, wild type.