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In order to determine whether the lack of Sec incorporation activity observed for the chimeric proteins was due to a lack of SECIS element binding, we tested whether or not they could bind the GPX4 SECIS element by electrophoretic mobility shift assay (EMSA).
To facilitate identification of KIF1C sequences needed for Rab6A binding, we tested whether the KIF1A motor domain binds to Rab6A. Figure 4A,C show that Rab6A bound to the KIF1A motor domain much more weakly (if at all), compared with KIF1C.
Given that the CTR of HupBMtb imparts upon it increased avidity with respect to DNA binding we tested whether the said region could protect DNA from enzymatic or non-enzymatic DNA strand breakage.
To more directly test whether L545 is involved in substrate binding, we tested whether MTSES-labeling of L545C could be protected by the presence of substrate using maleimide biotinylation.
Concerning DNA binding, we tested whether S/A or S/D mutation of the N- and C-terminal serines would have an effect on binding to the C-box consensus motif as it was shown for S160, 164, and 168 in the DNA binding domain (Kirchler et al., 2010, The role of phosphorylatable serine residues in the DNA-binding domain of Arabidopsis bZIP transcription factors).
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As phosphorylation of the BH3 proteins Bim and Bad is known to modulate binding to anti-apoptotic binding partners, we tested whether mutation of serine 10 altered the ability of Puma to bind Bcl-2, Bcl-xL and Mcl-1, thereby explaining the difference in apoptotic potential between this mutant and the WT protein.
Because VIG and Vig2 contain RNA binding domains, we tested whether these interactions with HP1 were dependent on an RNA component.
To confirm the removal of the neurospecific binding domain, we tested whether BoNT/BΔHC could enter and intoxicate hippocampal neurons.
Since the BNAR construct displays the same binding specificity as Roc1b, we tested whether the BNAR chimera could rescue the male sterility caused by the Roc1bdc3 null mutation (Fig. 5)[25].
Since OLA1 binds to the C-terminal of HSP70, the same binding site as for CHIP, we tested whether OLA1 and CHIP compete for binding to HSP70.
After confirming the Anxa5-GPI-enhanced binding of apoptotic cells to phagocytes, we tested whether the increased binding of apoptotic cells to phagocytes could enhance the ingestion of apoptotic cells.
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