1. MARCH6 and TRC8 degrade membrane‐associated mCherry‐CL1
MARCH6 and TRC8 degrade membrane‐associated mCherry‐CL1 AConfocal microscopy of mCherry‐CL1 HeLa cells treated with 20 nM bortezomib for 16 h. A KDEL antibody was used as an ER marker. Scale bar, 10 μm.B, CMembrane fractionation studies for mCherry‐CL1 levels in HeLa mCherry‐CL1 cells with or without proteasome inhibition (20 nM bortezomib 16 h) (B), or in AUP1, UBE2G2, MARCH6 or TRC8 KO HeLa mCherry‐CL1 clones (C). Briefly, cells were lysed by ball‐bearing homogenisation in a sucrose buffer, and the supernatant was ultracentrifuged at 50,000 rpm for 1 h to obtain the cytosol (C) and membrane (M) fractions. Tubulin and calnexin were used as control for the cytosolic and membrane fractions, respectively.D–GEctopic expression of TRC8 (D, E) or MARCH6 (F, G) in combined MARCH6/TRC8 null cells. Catalytically inactive mutants (MARCH6 C9A‐HA and TRC8ΔRING‐HA) were also overexpressed in the MARCH6/TRC8 null cells. mCherry‐CL1 levels were measured by flow cytometry and gated for HA‐positive cells (black line) (D, F). Basal mCherry‐CL1 levels in the parent reporter cells (red) and combined MARCH6/TRC8 null cells (green) are shown. HA‐tagged overexpressed ligases were also visualised by immunoblot (E, G).
Sandra Stefanovic‐Barrett et al. EMBO Rep. 2018;embr
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