1. AUP, UBE2G2 and TRC8 facilitate the degradation of mCherry‐CL1 through a shared pathway
AUP, UBE2G2 and TRC8 facilitate the degradation of mCherry‐CL1 through a shared pathway HeLa mCherry‐CL1 cells (shaded red) were transiently transfected with Cas9 and combinations sgRNA targeting AUP1, UBE2G2 or TRC8, generating mixed KO populations of single genes or combinations (AUP1/UBE2G2, TRC8/AUP1 or TRC8/UBE2G2, shown in shaded blue). mCherry levels were measured by flow cytometry, and bortezomib (Btz) (20 nM 16 h) was used as control for mCherry‐CL1 stabilisation (brown line).HeLa mCherry‐CL1 cells were transiently transfected with Cas9 and sgRNA targeting MARCH6, or in combination with AUP1 or UBE2G2, generating mixed KO populations. mCherry levels were measured by flow cytometry, and bortezomib (20 nM 16 h) was used as control for mCherry‐CL1 stabilisation.An mCherry‐CL1 TRC8 null clone (shaded green) was transiently transfected with Cas9 and sgRNA targeting AUP1, UBE2G2, MARCH6 or the empty sgRNA vector (EV) as described (blue line). Basal mCherry‐CL1 levels are also shown (shaded red).mCherry‐CL1 AUP1 null cells (shaded green) were transiently transfected with sgRNA to UBE2G2, TRC8 or MARCH6 (blue line), and mCherry levels were measured by flow cytometry. Bortezomib (20 nM 16 h) was used as control for mCherry‐CL1 stabilisation (brown line).
Sandra Stefanovic‐Barrett et al. EMBO Rep. 2018;embr
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