The optimal annealing temperature for each assay was determined using gradient PCR and a temperature gradient of 55°C to 70°C in twelve discrete steps in a G-storm GS-1 thermocycler (Geneworks Technologies Pty Ltd, SA, Australia). The PCR thermal profile was as follows: 95°C 2 min, ...
reporter assay showed that both mutations resulted in a marked reduction of transactivation activity. Because their dimerization activity was found to be intact by the two-hybrid system, it was possible that they were dominant-negative to wild type activity. When co-expressed with wild type, both...
Genotyping was done in all groups for an intronic SNP in AICDA (rs2580874), using real-time PCR genotyping assay. The less frequent genotype of AICDA in IgAD patients was AA, seen in 10.5% of the patients, which was much lower than the 30.8% in CVID patients and 38.2% in the controls...
Main conclusions were confirmed in different assays (qRT-PCR assay, HI-related phenotypic characterization assay, pollen viability and germination assay, Subcellular localization assay, RNA-seq assay). All the experiments were repeated at least three times independently with similar results. Randomization ...
(v:v), and the hexane phase was left to evaporate for 48 h. The remaining cells in the plates were disrupted in 0.1 M NaOH for 24 h and an aliquot was taken for measurement of cellular protein using the Bradford protein assay (Bio-Rad). The content of cellular cholesterol was ...
Functional assays with a dual-luciferase reporter assay system revealed that the truncating ISL1 protein lost transcriptional activation on the verified target genes MEF2C and NKX2-5. Additionally, the variant nullified the synergistic transactivation between ISL1 and TBX5 as well as GATA4, two ...
Functional analysis using the cell-based assay receptor selection and amplification technology (R-SAT) revealed that three variants (AT1-G45R, AT1-F204S, and AT1-C289W) displayed altered responses to Ang II and other AT1 receptor agonists and antagonists. Agonist responses to Ang II were absent...
After in vitro functional assay, variants P52L and R368H showed marked reduction of activity, confirming their role as loss-of-function mutations similar to previously determined variants G61E, N203S, and G329V. In contrast, variants G168D, A443G, and A465V showed no relevant effects and...
The pellet can then be used for the crystalline cellulose assay. Add 1 mL Updegraff reagent (Acetic acid: nitric acid: water, 8:1:2 v/v) to the pellet and heat at 100 °C for 30 min. Centrifuge at 9600 × g for 15 min, discard supernatant. Wash the pellet with water...
Cells were lysed using ice-cold RIPA buffer (250 mM Tris, pH: 7.5; 150 mM NaCl; 1% NP-40; 0.5% Na deoxycholate; protease inhibitors P2714 [Sigma-Aldrich, USA]). The total protein concentration of cell lysates was determined using the BCA Protein assay Kit (Thermo Fisher Scientific,...