1. CL1 hydrophobicity determines the specificity for recognition by MARCH6 or TRC8
CL1 hydrophobicity determines the specificity for recognition by MARCH6 or TRC8 A, BHeLa cells stably expressing wild‐type mCherry‐CL1 or mCherry‐CL1 mutants with alanine (red) mutations of the bulky hydrophobic residues (blue) (A) were transiently transfected with Cas9 and sgRNA targeting MARCH6, TRC8 or both (B). Hydrophobicity (arbitrary units) generated using “helical wheel projection” created by Don Armstrong and Raphael Zidovetzki (Version Id: wheel.pl, v ‐10‐20 21:23:36 don Exp.). Helical wheel generated using HeliQuest [75] (A). mCherry‐CL1 wild‐type and mutant levels (red) were measured following sgRNA transfection for the ligases (blue) or after treatment with bortezomib (Btz) (brown line) (B).CMembrane fractionation studies for mCherry‐CL1 wild‐type or CL1 mutants (CL1 4A, 2A and 6A) as previously described.DHeLa cells stably expressing the mCherry‐CL1 K3A mutant were transiently transfected with Cas9 and sgRNA targeting MARCH6, TRC8 or both as described.
Sandra Stefanovic‐Barrett et al. EMBO Rep. 2018;embr
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