To analyze the potential functional impact of an RBP on their mRNA targets, we have applied a robust technique called tethering assay. In this method, the protein under study is attached to an mRNA reporter through an artificial RNA鈥損rotein interaction. Therefore, the functional activity of a...
To analyze the potential functional impact of an RBP on their mRNA targets, we have applied a robust technique called tethering assay. In this method, the protein under study is attached to an mRNA reporter through an artificial RNA–protein interaction. Therefore, the functional activity of a ...
a deletion of the β-propeller domain of core subunits such as Vps11 and Vps18 will affect both CORVET and HOPS in vivo. In this regard, we were surprised that the CORVET Vps18∆N mutant, in contrast to Vps11∆N, was largely inactive in the tethering assay (Fig.5D), whereas the...
Previous studies have shown the importance of Smc5/6 in enriching cohesin at DSBs17. Accordingly, we depleted Smc5 in our DSB end-tethering assay (Extended Data Fig.2c,e). At 4 h post-DSB induction, Smc5 depletion resulted in a significant increase in DSB end-separation (Fig.2fand E...
Using a plasmid-ligation assay, we found that NHEJ is at the same low level in all of the xrs2Δ derivatives (Figure S4A), indicating that restoration of end tethering is not sufficient for NHEJ. Previous studies have shown that interaction between the Xrs2 FHA domain and Lif1 is required...
Using a novel in vitro SV pull down assay, we reported that SVs bind to a fusion comprising the C-terminal distal third (C3, aa 2137-2357; Wong et al., 2013). Here we limit the site further to the last 58 aa, beyond the proline rich site, by the absence of SV capture by a ...
Although LEDGF/p75 is believed to act as a cellular cofactor of lentiviral integration by tethering integrase (IN) to chromatin, there is no good in vitro model to analyze this functionality. We designed an AlphaScreen assay to study how LEDGF/p75 modulates the interaction of human immunodeficienc...
Protein-Fragment Complementation Assay The Venus-YFP variant of PCA was used in this study to examine protein-protein interactions in live yeast. Unless otherwise stated, endogenous proteins were tagged in haploid yeast by the PCR method with either VF1 or VF2 in the BY4741 and Y7043 strains,...
Phagocytosis assay LR73 cells were transiently transfected with the indicated plasmids. One day after transfection, the cells were incubated with TAMRA or CypHer5E or TAMRA and CypHer5E-stained apoptotic thymocytes, 2 μm carboxylate-modified red fluorescent beads or fluorescein-conjugatedE. colifor...
suggesting it may act downstream from budding. In a vesicle tethering assay, capture of vesicles by golgin-245 was substantially reduced in WDR11-knockout cells. Moreover, structured illumination microscopy and relocation assays indicate that the WDR11 complex is initially recruited onto vesicles rathe...