This chapter describes technologies that can be used to develop robust assays for screening compounds, more specifically the use of Homogeneous Time-Resolved Fluorescence Assay (HTRF) for IP3 measurement. A sample preparation protocol, parameters for assay optimization and examples of data analysis are ...
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Lastly, the new assay data correlated better with agonist-induced calcium responses in FLIPR ([R.sup.2] = 0.78) than with values determined by radioactive [IP.sub.1] method ([R.sup.2] = 0.64). Our study shows that the HTRF FRET-based assay detects [...
aInteraction between IPI7 and IPA1 in the yeast two-hybrid assay. IPA1 protein was fused with the GAL4 binding domain to generate BD-IPA1, and IPI7 with the GAL4 activation domain to form AD-IPI7. Blue clones in an X-Gal assay and clones grown on the SD-L-T-U medium indicate p...
Upregulation PAXIP1-AS1 was induced by CBP-mediated H3K27 acetylation (H3K27ac) via bioinformatic analysis and ChIP assay. Furthermore, PAXIP1-AS1 served as a competing endogenous RNA (ceRNA) to regulate PCBP2 expression by sponging microRNA-6744-5p (miR-6744-5p). Restoration experiments ...
Protein Phosphatase Assay Epitope-tagged Glc7p from KT1207 and KT1208 cells was immunoprecipitated according to the procedures described by Stuart et al. 1994, except that 100 ml of cells were grown, and after spinning down broken cell debris, the total supernatant was subjected to IP. Immunopre...
To assay behavioural flexibility, we utilised a touch screen-based appetitive reversal learning task in a separate cohort of experimental rats (WTn = 7,Cyfip1+/−n = 10). The reversal learning task first allowed an assessment of basic appetitive learning where rats had to learn that...
Yeast Two-Hybrid assay using full-length AQP2 as bait identified NEDD4 family interacting protein 2 (NDFIP2). NDFIP2 and its homologue NDFIP1 are adaptor proteins, which bind NEDD4 ubiquitin ligases via their PY-motifs and bring them close to target proteins. In HEK293 cells, NDFIP1/2...
The DTH response was monitored using the ear swelling assay. In this model, maximal swelling to HSV-1 antigen occurs at 24 h. Prior studies have established that animals are DTH responsive 5 days post immunization and remain sensitized for weeks (Lausch et al., 1985). DTH was elicited by ...
(E) and immunofluorescence assay (F) after indicated alteration of AURKAIP1 in TNBC cells. RT-qPCR (G) and western blots (H) were used to examine that whether DDX5 overexpression have an effect on AURKAIP1 expression in the indicated cells. The DDX5-rescued CCK8 (I) and transwell ...