For 2 h. Alternatively, the cell Macitentan lysates were incubated in 30 L anti-FLAG > 자유게시판

For 2 h. Alternatively, the cell lysates were incubated in 30 L anti-FLAG affinity agarose gel (50 slurry) at 4 overnight. Immunoprecipitates were washed with ice-cold RIPA buffer (400 L) for four times, resuspended in 50 l RIPA buffer and 10 l 6?sample buffer and then boiled for 5 min. G14 and FLAG-TPR1 proteins in the immunoprecipitates were analyzed by Western blots.Construction of chimeraschimera was constructed using 14z173 as the DNA template for the forward half-product and 203z14 as the DNA template for the backward half-product. 14243 was constructed using 203z14 as the template for the forward half product and Gz as the template for the backward half product. Its mirror image z243 was constructed using 14z151 as the template for the forward half product and G14 for the backward half product. Finally, 14DEF was constructed using 131z14 as the DNA template for the forward half-product and Gz as template for the backward half-product. Its mirror image zDEF was constructed using 14z224 as the DNA template for the forward half-product and G14 as template for the backward half-product. Primers for chimera construction are listed in Table 2. All G chimeras were checked by restriction mapping and then subcloned into pcDNA3 at HindIII and XbaI sites. The constructs were confirmed by dideoxynucleotide sequencing using Applied PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/12711626 Biosystem Big Dye Terminator v3.1 Cycle Sequencing Kits (Foster City, CA, USA).Inositol Phosphates (IP3) accumulation assayG chimeras were constructed from cDNAs encoding human G14 and Gz by using polymerase chain reaction (PCR) techniques. The N-terminal 37, 131, 182 and 203 residues of G14 were substituted by the corresponding amino acids of Gz to generate zN, 131z14, 182z14 and 203z14 chimeras, respectively. Primers were designed to produce two half-length fragments with overlapping regions; the forward fragment was generated with the antisense and T7 primers, whereas the backward fragment was made with the sense and reverse primers which target a BGH polyadenylation signal (BGH primers). The two half-products were then annealed together to generate a full-length fragment by another round of PCR using T7 and BGH primers. Mirror images of these constructs were generated analogously and were named 14N, 14z224, 14z173 and 14z151 chimeras. PCR (30 cycles each with 94 for 60 s, 58 for 60 s and 72 for 90 s) was carried out using AccuPrime PCR mix. The PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/8627573 1423 chimera was constructed using 182z14 as the DNA template for the forward half-product and 14z151 DNA template for the backward half-product. The zHEK293 cells were seeded on a 12-well plate at 2 ?105 cells/well one day prior to transfection. Cells were then transfected with 200 ng G using 2 L PLUS and Lipofectamine reagents in Opti-MEM. On the next day, cells were labeled with inositol-free Dubecco's modified Eagle's medium (DMEM; 750 L) containing 5 FBS and 2.5 Ci/mL myo-[3H]inositol overnight. Labeled cells were washed twice with the inositol phosphates assay medium (DMEM buffered with 20 mM HEPES, pH 7.5 and 5 mM LiCl) and were incubated for 1 h at 37 . Reactions were stopped by replacing the assay medium with 750 L ice-cold 20 mM formic acid and the lysates were kept in 4 for 30 min before the separation of [3H]IP from other labeled species by sequential ion-exchange chromatography as described previously [63].cAMP accumulation assayHEK293 cells were labeled overnight with [3H]adenine (1 Ci/ml) in culture medium containing 1 FBS. The labeled c.