13 Results
for author "Yue-wen Wang"
Figure 1.—SUMO homologs in C. reinhardtii: amino acid sequence alignment of yeast, human, and Arabidopsis SUMOs against SUMO homologs of C. reinhardtii.- TABLE 1SUMO proteins in C. reinhardtii and the extent of their similarity to their closest A. thaliana, S. cerevisiae, and human homologs, as well as scores for our SUMO-domain hidden Markov model
- TABLE 2Cleavage and binding domains of the C. reinhardtii SUMO proteins, their chromosomal localization, and their relative abundance in RT–PCR experiments (see also Figure 3)
Figure 2.—Predicted 3-D structure of human SUMO1 and CrSUMO96.
Figure 3.—Relative abundance of C. reinhardtii CrSUMO transcripts. Expression of the candidate SUMO genes was analyzed by quantitative real-time reverse transcription–PCR for expression in CC-503 cells at 25° and 42°. Each 25-μl reaction mixture contained cDNA equivalent to 50 ng of total input mRNA. Relative abundance was calculated with efficiency-corrected ΔCt values. Each data point is the average of an experimental triplicate and represents an individual trial.
Figure 4.—Immunodetection of C. reinhardtii CrSUMO96, CrSUMO148, and CrSUMO-like89A with anti-CrSUMOs and anti-AtSUMO-1 antibodies. Overexpression and purification of 6× His-tagged CrSUMOs from E. coli, antisera production, and immunoblot detection are shown. CrSUMO96 overexpressed in E. coli and affinity purified on a 6× His column (lanes 2, 5, 8, 11, 14, and 17), overexpressed and purified CrSUMO148 (lanes 3, 6, 9, 12, 15, and 18), and overexpressed and purified CrSUMO-like89A (lanes 4, 7, 10, 13, 16 and 19) were separated on 12% SDS–PAGE and stained with Coomassie blue (lanes 1–4) or detected after immunobloting using anti-6× His antibody (lanes 5–7), anti-AtSUMO1 antiserum (lanes 8–10), anti-CrSUMO96 antiserum (lanes 11–13), anti-CrSUMO148 antiserum (lanes 14–16), and anti-CrSUMO-like89A antiserum (lanes 17–19).
Figure 5.—Purification and identification of endogenous free CrSUMO96 from C. reinhardtii after boiling of cell extracts. Proteins in unboiled C. reinhardtii cell lysates (lanes 2 and 5) and boiled lysates at 1× (lanes 3 and 6) and 3× (lanes 4 and 7) loading concentrations were detected by Coomassie blue staining (lanes 1–4) or with anti-CrSUMO96 antiserum on immunoblots of the SDS–PAGE (lanes 5–7). The asterisk (*) indicates the endogenous free CrSUMO96. Molecular size markers are shown at the left (lane 1).
Figure 6.—Detection of endogenous SUMO-conjugated proteins from C. reinhardtii. Cell extracts were prepared in the presence or the absence of protease inhibitor cocktail or the isopeptidase inhibitor, NEM. Anti-CrSUMO96 antibody was utilized to detect the endogenous SUMO-conjugated proteins. The 26-kDa band indicated with an asterisk (*) is one of the nonspecific reacting proteins that can serve as a loading control.
Figure 7.—In situ localization of CrSUMO96 and its conjugated proteins by immunofluorescence. Wild-type C. reinhardtii cells grown in TAP media were stained with Sytox Green (top left) or detected with anti-CrSUMO96 antibody, which is recognized by the red fluorescent cyanine-5-conjugated goat anti-rabbit antibody (top middle). Images under transmitted light and confocal images are also shown in the top right and the bottom.