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Western blotting of cytoplasmic and nuclear fractions confirmed that the FAK-NLS mutant was indeed defective in nuclear localization.

To validate the SEC observations and to confirm that the oligomerization state of the triple mutant was indeed concentration-dependent, we turned to a combination of analytical ultracentrifugation and SEC-MALS.

Furthermore, to test that the slow development and low embryonic survival of the nrf-5 mutant was indeed due to the nrf-5 sa513 nrf-5 sa513 we took adVantage of the mutationtrain RB786 containing a large deletion in the gene stdh-1 located 0.09 c m awey from nrf-5 on chromosome V.

To directly validate whether the p53-R273H contact mutant was indeed capable of suppressing BMF expression and anoikis, and not because of the inherent anoikis resistance of the MDA-MB-468, HT29 or A431 cells, we performed a gene reconstitution experiment in the non-transformed MCF-10A cells.

The V419S sensor mutant was indeed able to interact with the ORAI1 R91W mutant, and this interaction was accompanied by a substantial decrease of FRET close to the plasma membrane, consistent with full-length STIM1 observations (Muik et al, 2008; Derler et al, 2009).

First, we determined the in vivo chromatin assembly profiles of two known in vitro chromatin assembly mutants, apc5 CA and rsp5 CA. We had already shown that the rsp5 CA mutant was indeed chromatin assembly defective in vivo (15), but surprisingly, the apc5 CA mutant was completely wild type in this assay, even when the 2μ plasmid was extracted from cells grown at 37°C (Fig. 3B).

Equilibrium and kinetic experiments demonstrate that folding of the ΔP174 mutant is indeed three-state.

If cell cycle progression in the FMP mutant is indeed altered, then we hypothesize that FMP43 protein is related to and somehow affects key regulatory mechanisms governing the cell cycle, as for instance the G1/S-phase control point.

Thus, to further validate that wildtype and the SPI-1 mutant are indeed localized inside 3-D HT-29 cells, we performed a differential immunofluorescence microscopy staining technique that allows one to distinguish between extra- and intracellular bacteria [45], [46].

How did the authors determine that this mutant is indeed nucleotide-free when purified?

As shown in Figure 2B, immunoblotting confirmed that the T51A mutant is indeed expressed and to the same extent as the WT PAGE4.