L., Cappellacci L., Franchetti P., Grifantini M., La Colla P., Loi A. TaqMan? gene expression assays used for CES1, CatA, and Hint1 were Hs00275607_ml, Hs00264902_m1, and Hs00602163_m1, respectively (Applied Biosystems, Foster City, CA). 18 S rRNA was used as an endogenous control. All experiments were performed in triplicate. Relative mRNA levels were calculated according to the method and expressed Benazepril HCl as fold change relative to the HHPC-1 sample for the CatA/CES1 experiment and relative to the clone A sample for the Hint1 experiment based on the manufacturer’s protocols (Applied Biosystems, Foster City, CA). Western Blot Analysis Protein extracts were prepared by incubating cells with preheated 1 NuPAGE? LDS sample buffer (Invitrogen) at 65 C for 10 min. Equivalent amounts of protein extracts were loaded into each well of a 10% BisTris NuPAGE polyacrylamide gel and resolved by electrophoresis using a NuPAGE MOPS running buffer (Invitrogen). Proteins were transferred to a nitrocellulose membrane using an iBlotTM gel transfer device (Invitrogen). Blotting and antibody incubation were performed using the Snap ID protein detection system (Millipore, Billerica, MA) according to the manufacturer’s protocol. Nitrocellulose membranes were blotted with SuperBlock T20 PBS blocking buffer (Thermo Scientific, Rockford, IL). Mouse monoclonal CatA, goat polyclonal CES1, and rabbit polyclonal Hint1 antibodies were diluted in blocking buffer, and the corresponding HRP-conjugated secondary antibodies were used. Blots were developed with SuperSignal West Dura extended duration substrate (Thermo Scientific), and the signal was detected using Gel Logic 2200 imaging system (Eastman Kodak). Cloning, Expression, RGS14 and Purification of Hint1 and Hint3 Human histidine triad nucleotide-binding proteins 1 and 3 (Hint1 and Hint3) were amplified from human fetal poly(A)+ RNA (Clontech) and ligated Benazepril HCl into pET17b made up of a pre-inserted N-terminal FLAG (DYKDDDK) affinity-tagged dihydrofolate reductase gene followed by a PreScission protease cleavable linker site (PS) (GE Healthcare) (15, 16). Human fetal poly(A)+ RNA was reverse-transcribed using a Superscript III first strand synthesis kit (Invitrogen) with random hexamers. Hint1 was amplified with forward primer 5-GGG GCC AAG CTT TGC AGA TGA GAT TGC CAA GGC TCA GG and reverse primer 5-CTA GTG GAT CCT TAA CCA GGA GGC CAA TGC ATT TGC CGA C. Hint3 was amplified with forward primer 5-GGG GCC AAG CTT TGC GGA GGA ACA GGT GAA CCG CAG and reverse primer 5-CTA GTG GAT CCT CAT GTT CTT AGT TTT TCA ATC AAG TGA TCA GCT GTG. Forward primers contained HindIII sites, and reverse primers contained BamHI sites for insertion into the pET17b-FLAG-ecDHFR-PS vector. The constructs were transformed into BL21-gold (DE3) (Stratagene, La Jolla, Benazepril HCl CA), and the cells were produced at 37 C in LB medium made up of 0.4% glucose and 100 g/ml ampicillin until the absorbance at 600 nm reached 0.4, at which point IPTG (0.5 mm) was added. Cells were harvested after 2.5 h growth at 37 C. The cell pellets were suspended in lysis buffer A (50 mm Tris/HCl, pH 8.0, 5 mm EDTA, 1 mg/ml lysozyme, and 50 g/ml NaN3) at a ratio of 25 ml/g of cells. After 5 min of incubation at room temperature, lysis buffer B (1.5 m NaCl, 0.1 m CaCl2, 0.1 m MgCl2, 20 g/ml DNase I, and 1 mm PMSF) was added at 2.5 ml/g cells (1 ml of lysis buffer B/10 ml of cell suspension). The lysate was incubated at room temperature for an additional 5 min followed by the addition of DTT (5 mm). The cell-free extract was collected by centrifugation at 15,000 rpm for 30 min at 4 C. The supernatant was loaded onto a 1-ml methotrexate-agarose column (Sigma), and the column was washed with 100 ml of buffer C (20 mm Tris/HCl, pH 7.0, 1 mm EDTA, and 1 mm DTT), followed by 600 ml of buffer D (1 m NaCl in buffer C). The fusion protein was eluted with buffer E (buffer D made up of 150 m trimethoprim (Sigma)). All fractions were analyzed by 10% SDS-PAGE (Invitrogen), and fractions made up of the fusion protein were combined. The salt concentration was reduced by diluting the protein solution with buffer C (7-fold). The dihydrofolate reductase tag was removed by treating with PreScission protease (10 units/mg, GE Healthcare). To run anion Benazepril HCl exchange column chromatography, the salt concentration of the protein solution was further reduced to 10 mm NaCl by concentrating the protein using an Amicon Ultra-4 centrifugal filter unit with an Ultracel-10 membrane from Millipore (Billerica, MA) and diluting the solution with buffer C. The protein was applied to a Mono Q 5/50GL column (GE Healthcare). The target protein was eluted in the flow-through with buffer C, Benazepril HCl although dihydrofolate reductase and proteases were.