Atches of 100 size-matched islets have been washed as soon as in serum-free RPMI medium and cultured in noncoated 6-well plates (Falcon) for 48 h. For protease inhibition studies, the islets were incubated in 100 nmol/liter CpdJ and DMSO, respectively. MS Loss- and Gain-of-Function Assays in MIN6 Cells–Stable knockdown of BACE2 and BACE1 in MIN6 cells was achieved working with lentiviral-mediated shRNA interference as outlined by common procedures from the RNAi Consortium (protocols are accessible online). Two different shRNA lentiviral plasmids (pLKO.1-puro) per protease, obtained as frozen bacterial glycerol stocks (Sigma-Aldrich), have been utilized (supplemental Table S1). Loss-of-function (LOF) cells lines and handle cell lines expressing the pLKO.1-puro non-mammalian shRNA manage plasmid were selected with 2 g/ml puromycin dihydrochloride (Sigma-Aldrich) and were used for experiments following 3 to 5 passages after infection. For gain-of-function (GOF) cell lines MIN6 cells have been electroporated with 5 g of DNA of the corresponding vector in AMAXA nucleofector V solution applying the AMAXA Nucleofector device and plan G-016 for MIN6 cells (Lonza Group Ltd). Cells were selected for three weeks with 380 g/ml G418 sulfate (Invitrogen). N-Linked glycopeptide capture of MIN6 cells was performed as described previously (9). For N-glycocapture of supernatants, LOF and GOF cell lines (eight 145 20 mm dishes per sample) were washed 3 times with PBS and incubated in phenol red-free OptiMEM (Invitrogen) for 24 h. The medium was collected and centrifuged at 200 g to eliminate cell debris. The supernatant was concentrated 240-fold employing Centricon plus-70 centrifugal filter units having a five,000-molecular weight cutoff (Millipore) and purified by 5 washing methods with two mol/liter urea, 0.1 mol/liter ammonium bicarbonate buffer.1-Oleoyl lysophosphatidic acid (sodium) Non-glycocapture experiments had been performed as described previously (eight).Nitro blue tetrazolium chloride JOURNAL OF BIOLOGICAL CHEMISTRYEXPERIMENTAL PROCEDURES Antibodies and Reagents–The following antibodies had been applied: BACE1 (Epitomics; 2882-1), BACE2 (Santa Cruz; sc10049), the BACE2 antibody employed in immunofluorescence staining was kindly provided by Roche, -Tubulin (Sigma; T6557), Glucagon (Millipore; 403101F), Insulin (SigmaAldrich; I8510), Pancreatic polypeptide (Sigma-Aldrich; SAB2500747), SEZ6L (R D; AF4804), SEZ6L2 (R D; AF4916), Somatostatin (Dako; A0566), Synaptophysin (Millipore; MAB5258), antibodies against the shed N-terminal TMEM27 ectodomain and full-length TMEM27 have been as previously described (15), V5 (Invitrogen; R960-25) and VAMP2 (Synaptic Systems, 104 211).PMID:24065671 Compound J (CpdJ) was synthesized byAPRIL 12, 2013 VOLUME 288 NUMBERDiscovery of -Secretase Substrates in -CellsLC-MS/MS Analysis–Peptides had been identified and quantified on a LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific) essentially as described (16). Full-scan MS spectra were acquired having a resolution of 60,000 within the Orbitrap analyzer. For each and every complete scan, the 5 most intense ions had been sequentially chosen and fragmented within the linear ion trap. Raw data had been processed and searched against a decoy database (consisting of forward and reverse protein sequences) of your predicted proteome from Mus Musculus (SwissProt, Version 57.12, in total 33,182 protein sequences) applying the SORCERERSEQUEST v4.0.three algorithm (17). The database search benefits had been further processed making use of the PeptideProphet (18) and ProteinProphet (19) applications along with the peptide false discovery price (FDR) was set to 1 on the.