Fig. 7
BRAF(C3A)-overexpressing mutant causes defects in membrane proteins’ vacuolar sorting. a Immunoblot analysis of FREE1 membrane association in WT and DEX::BRAF(C3A)-YFP plants after DEX induction. The FREE1 intensity was normalized by the loading control of anti-VSR as cell membrane (CM) fraction and anti-cFBPase as cell soluble (CS) fraction, and the CM/CS ratio of FREE1 in indicated genotype seedlings was quantified on the right. Error bars are the S.D. from three independent experiments. **P < 0.01 in Student’s t-test. b Electron micrographs of ILV formation in MVBs. Ultrathin sections were prepared from HPF/FS samples of indicated genotype plant roots without (−) or with (+) DEX induction, followed by immunogold labeling using VSR antibodies. The number of ILVs per MVB was statistically analyzed on 40 MVBs recognized by VSR antibodies. Error bars are the S.D. **P < 0.01 in Student’s t-test. Scale bar, 100 nm. c Immunoblots of membrane protein extracts from 7-day-old seedlings using anti-UBQ or anti-ManI. Note the accumulation of ubiquitin conjugates in DEX::BRAF(C3A)-YFP under DEX induction. d Complementation of sof524 with BRAFpro::BRAF(C3A) or BRAFpro::BRAF(C3A,A330V). The T1 generation-transformed seedlings were screened on MS with antibiotics for 5 days, and the seedlings were then transferred to MS medium without (−) or with (+) DEX for 7 days. Greening rate of seedlings after DEX treatment was quantified on the right. Error bars are the S.D. from three independent experiments. Scale bar, 1 cm
