Therefore, miR-221 is considered to be an attractive target for selective treatment against cancer

the HECT E3 Ubiquitin Ligases increased polyubiquitination of Glis3 and promoted Glis3 proteolytic degradation. Collectively, our study identifies PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19741130 the E3 ubiquitin ligase, Itch, as a critical regulator of Glis3-mediated transcription by controlling the level of Glis3 protein. Our results suggest that Itch functions as a novel modulator of Glis3-mediated transcriptional regulation and as such might modulate Glis3 target genes in cells in which it plays a critical role, such as the regulation of pancreatic beta cell generation and insulin gene expression as well as in Glis3-associated diseases, such as type 1 and 2 diabetes, and polycystic kidney disease. Materials and Methods Cells and Growth Conditions Rat insulinoma INS-1832/13 cells, kindly provided by Dr. H. Hohmeier, were maintained in RPMI 1640 supplemented with 10% fetal calf serum, 10 mM HEPES, 2 mM glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin, 100 g/ml streptomycin, and 50 M -mercaptoethanol. HEK293T cells were purchased from ATCC and cultured in DMEM containing 10% FBS supplemented with 10% FBS. FreeStyle 293-F cells were obtained from Life Technologies and grown in FreeStyle 293 Expression Medium. Gel-enhanced Liquid Chromatography Mass Spectrometry In-gel digestion and mass spectrometry were performed essentially as described previously. Briefly, immuno-precipitated proteins were separated by SDS-PAGE and the lanes were digested with trypsin for 8 hours using a Progest robotic digester. The resulting peptides were extracted by a series of washes with water, acetonitrile, and formic acid. All supernatants were pooled during the collection process and were lyophilized to dryness. The lyophilized samples were resuspended in 40 L of 0.1% formic acid. NanoLC-ESI-MS/MS analyses were then performed using an Agilent 1100 nanoLC system on-line with an Agilent XCT Ultra ion trap mass spectrometer with the Chip Cube Interface. 20 l of the peptide mixture were loaded onto an Agilent C18 chip followed by a 15 minute wash of 5% acetonitrile, 0.1% formic acid. Peptides were eluted by applying a linear acetonitrile gradient over 45 minutes. This was followed by a 5 minute acetonitrile gradient and then a 10 minute hold at 95% acetonitrile, 0.1% formic acid. A peak list was generated from the data obtained from the nanoLC-ESI-MS/MS analysis using the Data Extractor feature of the Spectrum Mill software from Agilent. The Data Extractor settings included limiting the data search to deconvolved ions observed between 400 PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19741685 and 5000 Da and a retention time between 10 minutes and 50 minutes. The resulting extracted data were then searched against the NCBI human and rodent species limited database using the MS/MS Search function in the Spectrum Mill software. Proteins identified with a distinct summed MS/MS search score greater than 17 were tabulated. At this threshold, the false positive rate is approximately 13% as determined by searching against a reversed sequence database. Yeast-two-hybrid Analysis Yeast-two-hybrid analysis was performed by Hybrigenics Inc.. The coding sequence for the Glis3 amino terminus was PCR-amplified and cloned into pB27 to encode a Glis3-LexA fusion protein. The resulting construct was used as bait to TL32711 screen a murine pancreatic beta cell library constructed in plasmid p6. The mated bait and prey strains were spread on medium lacking histidine, leucine, and tryptophan and supplemented with 5.0 mM 3-aminotriazole to prevent bait auto-activation. 179 clones were