Because no loss-of-function mutations in the 3 gene could be found in the MMIH patients examined (Lev-Lehman et al., 2001), other protein deficits could underlie the syndrome. UBXD4 led to a significant reduction of 3* nAChRs in rat cortical neurons and dPC12 cells. Biochemical and immunofluorescence studies of endogenous UBXD4 showed that this protein is located in both the ER and experiments with charged Ni+ columns showed that this 3 subunit can bind to (His)6-TYG-tagged UBXD4 protein expressed in HEK293 cells. Turbo polymerase (Stratagene). The cDNA sequence corresponding to the large cytoplasmic domain name (amino acids 305C490) of the mouse 3 subunit was amplified using the forward primer 5-GGA ATT CCA TAT GCT CCT CTT CAC TAT GAT TTT TGT CAC-3 and the reverse primer 5-ACG CGT CGA CCA GAA ATA ATC CTG CAG TTC CTA AAA TG-3 by PCR and subcloned into the 0.001). indicated that 3 and 2 levels increased 2.2-fold while 4 levels increased 1.4-fold compared with control. For pull-down experiments, HEK293 cells stably expressing nAChRs were transfected with (His)6-TYG-tagged UBXD4. Cells lysates were diluted with 1 ml of wash buffer (0.5 m NaCl, 50 mm imidazole, 20 mm Tris, pH 7.0) and loaded onto Ni-NTA agarose (Qiagen) at 4C, and then incubated overnight. Proteins were eluted with elution buffer (0.5 m NaCl, 0.2 m imidazole, 20 MC-Val-Cit-PAB-rifabutin mm Tris, pH 7.0). Each eluted sample was concentrated MC-Val-Cit-PAB-rifabutin with a microsep centrifugal concentrator (Pall Life Sciences) to give final volumes of 20 l. Eluted samples were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Bound proteins were detected by Western blotting using Supersignal West Femto (Pierce Biotechnology). p62 pull-down assays and immunoprecipitation. Pull-down assays were conducted with the agarose-immobilized p62-derived UBA domain name (Biomol) as previously explained (Rezvani et al., 2007). Immunoprecipitations were conducted as explained previously (Wiser and Schweiger, 1986; Karan et al., 2005). For the IP experiment shown in Figures 1and ?and33 0.05). MC-Val-Cit-PAB-rifabutin The brackets on the right (test for normally distributed variables to conclude the significant differences between control and subjects or by one-way ANOVA and Tukey multiple comparison post assessments when appropriate. A value 0.05 was considered statistically significant. All data are reported as imply SEM. Results A UBX-containing protein binds to 3* nAChRs To identify proteins involved in the trafficking of 3* nAChRs to the PM, we performed Y2H screen against a mouse brain cDNA library. cDNA encoding the large intracellular domain of the 3 nAChR subunit and the flanking transmembrane (TM) regions 3 and 4 (amino acids 305C490) was used as bait. The TM regions were included in the bait because intracellular domains can adopt a more precise conformation when expressed with TM regions on either end (Bedford et al., 2001). The Y2H screen yielded an conversation between the large intracellular loop of the 3 nAChR subunit and a novel protein. Sequencing deciphered a 589 nt reading frame corresponding to the truncated N terminus of the UBXD4 protein (Fig. 1eyes closed gene and vertebrate p47) is usually a reversible competitive inhibitor of MC-Val-Cit-PAB-rifabutin cathepsin L (Chu et al., 1995; Soukenik et al., 2004); UBX (ubiquitin regulatory X), consisting of 80 amino acid residues, can typically be found at the C terminus of a variety of eukaryotic proteins of differing functions (Buchberger et al., MC-Val-Cit-PAB-rifabutin 2001; Buchberger, 2002; Bogan et al., 2003; Schuberth and Buchberger, 2008). A search in GenBank revealed sequences of the human (accession number: “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_181713″,”term_id”:”1519473737″NM_181713) and rat homologs (accession number: XR_007472) to UBXD4. Comparison of all three sequences indicated that UBXD4 is usually highly conserved between the three species (85.7%, 95.5%, and 86.0% identical between mouse and human, mouse and rat, and rat and human, respectively), suggesting that it may possess an important function in mammals. To examine the binding specificity of UBXD4 for other nAChR subunits, the intracellular loop plus the respective TM flanking regions of 4, Rabbit Polyclonal to SIRT3 7, 2, and 4 were used as probes in additional Y2H screens against the original UBXD4 clone (clone 42). Clone 42 bound to the intracellular loop of both the 3 and 4 nAChR subunits, but not to the corresponding loops of 7, 2, and 4 (Fig..