Journal: The Plant Journal

Article Title: Arabidopsis peroxisomes possess functionally redundant membrane and matrix isoforms of monodehydroascorbate reductase

doi: 10.1111/j.1365-313x.2005.02503.x

Figure Lengend Snippet: Figure 2. Suborganellar distribution, membrane association, and topologi- cal orientation of MDAR polypeptides in Arabidopsis peroxisomes. Proteins in Arabidopsis suspension cell peroxisomes (pooled from three sucrose gradients, e.g. Figure 1) and peroxisomal subfractions were precipi- tated in trichloroacetate, subjected to SDS-PAGE (50 lg protein per lane), and then analyzed on blots for MDAR polypeptides that were detected with anti- cucumber MDAR antibodies. Replicate immunoblots probed with anti-APX or anti-catalase IgGs provided positive controls for membrane- and matrix- localized proteins, respectively. (a) Membrane association. Peroxisomes (lane 1) were subjected to hypotonic burst in 25 mM HEPES-KOH (pH 7.5) and centrifuged to produce water- solubilized protein supernatants (lane 2) and water-insoluble membrane pellets. Peripheral membrane proteins were extracted from these pellets in 0.2 M KCl and extracts were centrifuged to generate a KCl-soluble supernatant (lane 3) and KCl-insoluble pellet (lane 4). Cytosolic fractions (cleared of membranes by centrifugation, lane 5) and clarified homogenate (CH) (sample applied to the gradients, lane 6) also were examined. (b) Membrane topology. Intact peroxisomes (lane 1) were treated with proteinase K without (-) and with (þ) presolubilization in Triton X-100 (lanes 2 and 3, respectively).

Article Snippet: Primary and secondary antibodies were used as follows: rabbit anti-cucumber 47-kDa MDAR antiserum (1:1000) (Sano et al., 1995), rabbit anti-cucumber peroxisomal APX IgGs (1:1000) (Corpas et al., 1994), rabbit anti-cottonseed catalase IgGs (1:1000 or 1:2000) (Kunce et al., 1988) and goat anti-rabbit alkaline phosphatase conjugate (1:10 000) (Bio-Rad).

Techniques: Membrane, Suspension, SDS Page, Western Blot, Centrifugation

Journal: The Plant Journal

Article Title: Arabidopsis peroxisomes possess functionally redundant membrane and matrix isoforms of monodehydroascorbate reductase

doi: 10.1111/j.1365-313x.2005.02503.x

Figure Lengend Snippet: Figure 5. In vivo immunofluorescence sorting analyses of an Arabidopsis approximately 54-kDa MDAR polypeptide in Arabidopsis and BY-2 suspen- sion cells. Arabidopsis (a, b) and BY-2 (c, d) cells bombarded with a gene coding for myc- AtMDAR4 were allowed to express the transgene for 2.5 and 5 h, respectively. Cells then were fixed in formaldehyde, permeabilized in Triton X-100 and co- immunolabeled with anti-myc (a, c) and anti-catalase (b, d) primary and fluorescence dye-conjugated secondary antibodies. Solid arrows point to examples of co-localization between the two fluorescent markers within catalase-containing peroxisomes. All micrographs are confocal projection images. Bars in (a, c) ¼ 10 lm. Figure 6. In vivo topological orientation of AtMDAR4 expressed in Arabid- opsis cells. Cells bombarded with myc-AtMDAR4 or myc-AtMDAR2 were fixed in formaldehyde, perforated/digested with Pectinase and differentially perme- abilized (plasma membranes only) with digitonin. (a–d) Transformed cells co- labeled with anti-myc antibodies for expressed myc-AtMDAR4 (a) or myc- AtMDAR2 (c) and with anti-catalase IgGs for endogenous peroxisomal catalase (b, d). (e, f) Representative mock-transformed cells colabeled for cytosolic microtubules (anti-a-tubulin antibodies) (e) and peroxisomal cat- alase (f). Bar in (a) ¼ 10 lm.

Article Snippet: Primary and secondary antibodies were used as follows: rabbit anti-cucumber 47-kDa MDAR antiserum (1:1000) (Sano et al., 1995), rabbit anti-cucumber peroxisomal APX IgGs (1:1000) (Corpas et al., 1994), rabbit anti-cottonseed catalase IgGs (1:1000 or 1:2000) (Kunce et al., 1988) and goat anti-rabbit alkaline phosphatase conjugate (1:10 000) (Bio-Rad).

Techniques: In Vivo, Immunolabeling, Clinical Proteomics, Transformation Assay, Labeling