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At 24 h after assay initiation, the presence or absence of clumps at the opening of the dispenser was scored.
Cyanide concentration was measured at several positions in a parallel assay tube without nematodes at one and 24 h after assay initiation (Figure 2B).
By 5 h after assay initiation, nematodes could be seen to accumulate at both openings of the chemical dispensers with 2 mM KCN (Figure 1A), and in a region of about 5 mm diameter around the small opening of the dispenser with 5 mM KCN (Figure 1C).
One microlitre of test compound was added to each well on the day of assay initiation.
All plates and reagents were equilibrated to room temperature prior to assay initiation and all results were obtained using a Synergy II plate reader (BioTek).
U-87 MG spheroids were treated on the day of assay initiation while MDA-MB-231 spheroids were pretreated for 24 h and during the assay.
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Transcription of the yeast CYC1 promoter fused to a sequence lacking guanosine residues provided a rapid, sensitive assay of initiation by RNA polymerase II in yeast extracts.
Subsequently all fractions were used for CFU-F assay or initiation of in vitro MSC cultures.
Plasma concentrations of sVEGFR2 were measured by enzyme-linked immunosorbent assay before initiation of treatment with pazopanib (baseline) and at 4, 8, and 12 weeks of pazopanib treatment.
Disruption of the eRF3-PABP interaction inhibits translation and the addition eRF3 to in vitro translation assays stimulates initiation [ 59].
Abortive initiation assays were initiated using specific dinucleotide primers in the presence of only the next α-P-NTP (for AP3, 250 µM GpU + 50 µM α-P-UTP; for 23S, 250 µM UpG + 50 µM α-P-CTP, see Materials and methods).
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