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. 2015 Nov 23;211(4):775–784. doi: 10.1083/jcb.201504109

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© 2015 Ninagawa et al.

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Figure 4. — Effect of EDEM-TKO on mCD3-δ–ΔTM degradation. (A) Schematic structures of WT and two insertion mutants of mCD3-δ–ΔTM. (B) Immunofluorescence of HCT116 cells expressing transfected WT or insertion mutant of mCD3-δ–ΔTM using anti-HA. (C) PNGase sensitivity assay of WT and two insertion mutants of mCD3-δ–ΔTM expressed in HCT116 cells by transfection (immunoblotting with anti-HA). The asterisk denotes partially deglycosylated mCD3-δ–ΔTM. (D) Pulse-chase to determine the degradation rate of transfected WT mCD3-δ–ΔTM in WT and hEDEM-TKO cells using anti-HA (n = 3). (E) Pulse-chase to determine the degradation rate of transfected WT mCD3-δ–ΔTM in WT cells and those treated with 40 µM MG132 using anti-HA. This experiment was completed once. (F) Pulse-chase to determine the degradation rate of transfected insertion mutants of mCD3-δ–ΔTM in WT and hEDEM-TKO cells using anti-HA (n = 3). (D–F) Means ± SD are shown. (D and F) *, P < 0.05; **, P < 0.01.