The ecsA::intron mutant and wild-type Newman strain showed a similar pmf as monitored with the fluorescent dye diSC3 5) (data not shown), indicating that the pleitropic phenotype of the mutant is not an indirect effect of altered pmf.
Repeated trials of each of the mutants also established that the kinetics of oligomer formation were highly reproducible as monitored by increases in ThT fluorescence.
In fact, the transient transfection of PrP mutants lead to ∼40% of cell death after 72 h of transfection as monitored by the MTS assay (not shown).
Both wild-type TSC2 and the phosphorylation-site mutants blocked the constitutive, growth factor-independent mTORC1 activity in Tsc2-deficient cells, as monitored by S6K1 and S6 phosphoryation and 4E-BP1 mobility shifts indicative of phosphorylation status (Figure 5C).
Other phenotypes of hlh-25 (ok1710 ) animals included decreased movement as monitored by thrashing and locomotion assays, leading us to classify hlh-25 mutants as locomotion variants.
Indeed, not only was NLS binding retained by these mutants, but the affinity of binding to the CDC5L NLS3 (as well as to the Prp31 NLS) peptide, as monitored by ITC, was little affected by these amino acid changes; the small differences in binding affinity, such as the deletion mutant K1, might reflect a minor influence on the stability of the protein (Supplementary Fig. 5).
Ribosomes isolated from two mutants with the most pronounced phenotypic changes had increased affinities for aa-tRNA, and surprisingly, increased rates of peptidyltransfer as monitored by the puromycin assay.
The kaiBC promoter activity in each mutant strain was monitored as bioluminescence [38].
In order to address this possibility, Hhat transfected COS-1 cells were treated with cycloheximide, to block new protein synthesis, and levels of WT and Hhat truncation mutant proteins were monitored as a function of time.
Here, N chemical shifts for ubiquitin and ubiquitin mutants were monitored as a function of increasing dsk2 concentration.
