Production and Purification of Recombinant Proteins The following constructs for expression of glutathione S-transferase (GST) fusion proteins, namely, were used: GST-USP8542-1080 (kindly provided by Dr. E. Martegani, University of Milano-Bicocca, Italy), GST-USP81_438, and GST-USP81-m (kindly provided by Dr. S. Urbe, School of Biomedical Sciences, Liverpool, U.K.), and GST-USP8542–660. GST-USP8542_1Q80 fusion construct contains the second SH3-binding […]
Subcellular Protein Fractionation and Immunoblotting Analysis One-percent Triton X-100-soluble protein lysates and hypotonic protein extracts from freshly isolated spermatogenic cells were obtained as described. To separate cytosolic and membrane particulate fractions, the hypotonic extract from postnuclear supernatant was spun at 100 000 X g for 1 h at 4°C. The resulting supernatant is the cytosolic […]
Animals Testes were collected from 3- to 4-mo-old CD1 male mice (Charles River, Calco, Italy). Handling of mice and experimental procedures were reviewed and approved by the Ethical Committee of the University of Milano and were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals promulgated by the Italian Minister […]
The number and position of ubiquitin molecules bound to lysine residues of a target protein are, in fact, an important signal for directing the subcellular localization and intracellular traffic of the protein cargo, and the ubiquitin-specific proteases like USP8 are critical in defining the trimming of the ubiquitin moiety.
The deubiquitinating enzyme USP8 (ubiquitin-specific peptidase 8; previously named UBPy, ubiquitin-specific processing protease-y) was originally identified as a putative protein encoded by a cDNA expressed in human myeloblasts (hUBPy) and then found, through independent yeast two-hybrid screenings of mouse libraries, as a protein (mUBPy) that interacts with STAM2 (also known as Hbp) and the Ras-guanine […]