Anti UBE2J1  

Western blot using Rockland's affinity purified anti-Ube2j1 antibody shows detection of Ube2j1 in 293 cells over-expressing Myc-Ube2j1 (Lane 2). Lane 1 contains lysate from mock-transfected 293 cells. Personal Communication, A. Weissman & T. Shang, CCR-NCI, Frederick, MD

(A)？Sertoli cells were cultured and treated as described in？Materials and Methods？and the legend to？Fig. 1. Blots were probed with an anti-Ube2j1 antibody (see？Table 1) using actin which served as a protein loading control. TGF-β3, but not testosterone, was shown to induce the steady-state level of Ube2j1 in the Sertoli cell epithelium (a). Histogram shown in？b？represents composite results from several immunoblots (n？= 3？4), such as the one shown in？a. Each data point was normalized against actin, and time 0 in each experimental group was arbitrarily set at 1 against which statistical analysis was performed. *,？P<0.05.？(B)？Immunoblot analysis (7.5%？T？gel) using lysate from Sertoli cells (SC) (30 μg protein) and the corresponding rabbit (Rb) (Ba) or mouse (Ms) (Bb) anti-Ube2j1 antibody (see？Table 1) as well as the normal rabbit or mouse IgG, illustrating the specificity of the anti-Ube2j1 antibody. M, marker.？Fig 6. PMID:？20682309

(C)？Dual-labeled immunofluorescence analysis in？a-i？illustrating an increase in co-localization between occludin (green) and Ube2j1 (red) after TGF-β3, but not testosterone, treatment (Ci versus c,f). DAPI (blue) was used to visualize nuclei. White arrowheads in？Ci？indicate an increase in the co-localization of occludin with Ube2j1 after TGF-β3 treatment (Ci versus？Cc,f). Scale bar = 10 μm in？Ca, which also applies to？Cb-i.？ Fig 6. PMID:？20682309

(D)？The study shown in？(C)？was performed using claudin-11 (green), another putative TJ-protein at the BTB, with similar results. Scale bar = 10 μm in？Da, which also applies to？Db-i. Fig 6. PMID:？20682309

Most modifiers mature by proteolytic processing from inactive precursors (“a” = amino acid). Arrowheads point to the cleavage sites. Ubiquitin is expressed either as polyubiquitin or as a fusion with ribosomal proteins. Conjugation requires activating (E1) and conjugating (E2) enzymes that form thioesters (S) with the modifiers. Modification of cullins by RUB involves SCF(SKP1/cullin-1/F-box protein)/CBC(cullin-2/elonginB/elonginC)-like E3 enzymes that are also involved in ubiquitination. In contrast to ubiquitin, the UBLs do not seem to form multi-UBL chains. UCRP(ISG15) resembles two ubiquitin moieties linked head-to-tail. Whether HUB1 functions as a modifier is currently unclear. APG12 and URM1 are distinct from the other modifiers because they are unrelated in sequence to ubiquitin. (From Jentsch & Pyrowolakis (2000); see references below.)