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#0 dbbase_sql->halt(Invalid SQL: select * from kra_comment where pid='86632' and iffb='1' order by id limit 0,10) called at [E:\phpweb\phpwebsite\1011kra\63965966\includes\] #1 dbbase_sql->query(select * from {P}_comment where pid='86632' and iffb='1' order by id limit 0,10) called at [E:\phpweb\phpwebsite\1011kra\63965966\comment\module\CommentContent.php:167] #2 CommentContent() called at [E:\phpweb\phpwebsite\1011kra\63965966\includes\] #3 printpage() called at [E:\phpweb\phpwebsite\1011kra\63965966\comment\html\index.php:13] 留言点评--晋红兵
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(all Pan1green fluorescent protein[GFP]) strains have been generated by C-terminal
Cells ended up developed to early log mTOR Inhibitor 1 web section, shifted to 30 for 15 min, and incubated with -factor. For Ste3-GFP visualization, the pH of the society was modified with 10 mM Tris, pH seven.5, 5 min in advance of observation. The cells had been then pelleted and resuspended while in the identical media at two? OD/ml density and imaged. Ste3-GFP illustrations or photos were obtained with an LSM 510 META confocal microscope (Carl Zeiss MicroImaging,, Thornwood, NY) geared up along with the appropriate lasers and filter set.Fluorescence MicroscopyPan1-GFP photographs were gathered using an Axiovert 135TV inverted microscope (Carl Zeiss MicroImaging) using a Sensicam QE charge-coupled unit digicam (Cooke, Romulus, MI), Zeiss a hundred one.four nu.(all Pan1green fluorescent protein[GFP]) strains ended up produced by C-terminal chromosomal integration of polymerase chain response PubMed ID: (PCR) solutions as explained in Longtine et al. (1998). The parental quintuple deletion pressure (BWY2571) was generated by transformation using a PCR fragment that contains homologous untranslated areas of EDE1 bordering the G418-resistant cassette and choice for growth on G418 plates (Longtine et al., 1998). The plasmids employed in this review are detailed in Supplemental Table two. DNA manipulations were executed making use of normal strategies, employing both T4 DNA polymerase-mediated ligations in Escherichia coli or homologous recombination with overlapping DNA fragments followed by plasmid rescue in S. cerevisiae. Amino acid substitutions had been created working with QuikChange XL site-sate for your endocytic function on the yeast epsin C-termini in ENTH cells. Two other candidate adaptors will be the yeast proteins Yap1801 and Yap1802, the homologues from the mammalian endocytic clathrin assembly proteins CALM/AP180 (Wendland and Emr, 1998; Wendland et al., 1999). Like AP180/ Tranquil, Yap1801/2 have an AP180 amino(N)-terminal hoVol. 19, JulyL. Maldonado-Baez et al. ?directed mutagenesis kit (Stratagene, La Jolla, CA) in accordance to your manufacturer‘s guidelines. All restriction enzymes had been acquired from New England Biolabs (Ispwich, MA).Plasmid Shuffle, , 5 cells expressing ENT1 or ENT2 from a TRP1 or URA3 plasmid have been employed for counterselection on 5-fluoroanthranilic acid (5-FAA) or 5-FOA, respectively. Cells cotransformed with ENT1 mutant or truncation URA3 and WT TRP1 plasmids have been grown on 5-FAA plates (to evict the TRP1 plasmid) at 30 for 3 d. Alternatively, cells cotransformed with ENT1 mutant or truncation TRP1 and WT URA3 plasmids ended up developed on 5-FOA plates (to evict the URA3 plasmid) at 30 for three d.-Factor Uptake ExperimentsS- PubMed ID: -factor was purified and -factor internalization assays were done using a constant existence protocol as described formerly (Dulic et al., 1991). Cells had been developed to early log period, shifted to thirty for fifteen min, and incubated with -factor. Samples were being taken just after incubation within the indicated time and preserved in ice-cold phosphate buffer, pH 6.0 (40 mM KH2PO4 and 6.0 mM K2HPO4) or citrate buffer, pH one.0 (fifty mM Na3C6H5O7 2H2O). Samples were gathered on glass filters (VWR, West Chester, PA) and counted on an LS6500 scintillation counter (Beckman Coulter, Fullerton, CA).
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