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. 2022 Nov 23;11:e68253. doi: 10.7554/eLife.68253

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© 2022, Dingus et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 7. — (A) Examples of nanobody-TagBFP expression in 293T cells following transient transfection before (WT) and after (mut1) stage 1, conservation-based mutagenesis. Red signal is from co-transfected CAG-dsRed plasmid. (B) Examples of nanobody-TagBFP expression in 293T cells following transient transfection for nanobodies with Gly52. Wild-type, first-pass conservation-based mutants (mut1), and mutants with up to two additional mutations, Gly52Phe and X90Gln, are depicted. (C) Numbers of stable versus unstable nanobodies (75 total) following first-pass conservation-based mutagenesis (stage 1 mut) and final mutagenesis (+G52F+X90Q). (D) Example mutant variants tested for specific mutationally stabilized nanobodies in order to identify necessary stability mutations. (E) Examples of nanobody-TagBFP expression in 293T cells following transient transfection for nanobodies effectively stabilized by one to two mutations. Scale bar is 20 µm.