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Figure S1. RING domain of the Arabidopsis RING peroxins contains E3 ligase activity in different in vitro systems.(A) E3 ligase assays using rabbit E1, human UBCH5c (E2), bovine ubiquitin, and MBP-PEXRING proteins. UBCH5c seemed to promote the E3 activity of PEX12RING. MBP-PEX2RING was used in the minus E1 and minus E2 reactions. Arrowheads point to monoubiquitinated MBP-PEXRING. Asterisk indicates Ubs. (B, C) E3 ligase assays using yeast E1, AtUBC8, bovine ubiquitin, and MBP-PEXRING proteins. Asterisks indicate Ub-conjugated MBP-PEXRING proteins. Low molecular weight (MW) bands are mostly E2-Ub conjugates. Numbers to the left of the gels are MW markers in kDa.

Figure S2. Immunoblot analysis of fusion proteins extracted from yeast strains used for yeast two-hybrid (Y2H) assays shown in Figure 3B.  BD- and AD-fusion proteins were detected with anti-LexA (top panel) and anti-HA (bottom panel) antibodies, respectively. Numbers to the left of the gels are molecular weight (MW) markers in kDa.

Figure S3. Immunoblot analysis of fusion proteins extracted from yeast strains used for yeast two-hybrid (Y2H) assays shown in Figure 4B.  Anti-HA antibodies were used to detect AD-fusion proteins (RING proteins). Numbers to the left of the gels are molecular weight (MW) markers in kDa. Arrows indicate the expressed RING domains.

Figure S4. Subcellular localization of YFP-DSK2 in transgenic plants and identification of T-DNA insertion mutants of DSK2 genes.(A) Confocal images from Arabidopsis transgenic lines co-expressing YFP-DSK2 and the peroxisomal marker CFP-PTS1. Scale bar = 10 μm. (B) Gene structure of DSK2a and DSK2b. Positions of the T-DNA insertions are indicated. Black boxes are coding regions and white boxes are untranslated regions (UTRs). (C) Reverse transcription polymerase chain reaction analysis of homozygous T-DNA insertion lines for DSK2a and DSK2b. Ubiquitin 10 (UBQ10) served as a loading control.

Table S1. Primers used in this study

Table S2. Vectors used in this study

FilenameFormatSizeDescription
JIPB_12014_sm_FigS1.pdf77KSupporting info item
JIPB_12014_sm_FigS2.pdf58KSupporting info item
JIPB_12014_sm_FigS3.pdf34KSupporting info item
JIPB_12014_sm_FigS4.pdf56KSupporting info item
JIPB_12014_sm_Tables.doc111KSupporting info item

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