Figure 5

HRD1 accelerates ABCG8 degradation by reducing its protein stability through the inhibition of ABCG8 N-glycosylation.

(a), (b) Stability of WT- or N619Q-ABCG8 in HEK293 cells was determined by CHX (50 μM) chase experiment (a). HM or Non-G forms of ABCG8 were quantified and normalized by HSC70 expression. Data are presented as the percentage of the amount detected at 0 hr (b). **p < 0.01, ***p < 0.001, versus WT ABCG8-transfected cells; Student's t test (n = 3). (c) Steady-state expression of HEK293 cells transfected with WT- or N619Q-ABCG8. Cell lysates were subjected to immunoblotting (upper panel). Relative expression of Non-G form of ABCG8 were calculated (n = 3) (lower panel). (d), (e) Stabilities of HM and Non-G forms of WT-ABCG8 protein derived from Myc-HRD1 (1 μg)-transfected HEK293 cells were determined by CHX chase experiment (d). HM or Non-G forms of WT-ABCG8 were quantified (e). *p < 0.05, **p < 0.01, versus HM ABCG8 bands; Student's t test (n = 3). (f), (g) Stability of Non-G N619Q-ABCG8 protein in HEK293 cells transfected with Myc-HRD1 (f). Non-G N619Q-ABCG8 protein was quantified (n = 3) (g). (h–j) WT-ABCG8 and Myc-tagged WT- or C329S-HRD1 were co-transfected and cells are subjected to CHX chase experiment. After recovery, cell lysates were digested with (i) or without (h) EndoH and were analyzed by immunoblotting. Stability of total ABCG8 protein after transfection with HRD1 constructs was quantified using Endo H (+) shown in (i) and normalized by HSC70 expression (j). *p < 0.05, **p < 0.01, ^#p < 0.05, versus pcDNA6-transfected cells; Student's t test (n = 3). Student's t test (n = 3). (k) Steady-state expression of HEK293 cells transfected with si-GL2 (control si-RNA) or si-HRD1. Cell lysates were subjected to immunoblotting (upper panel). Relative expression of HM ABCG8 was calculated (n = 3) (lower panel). (l), (m) Stabilities of HM form of WT-ABCG8 protein derived from si-GL2 or si-HRD1-transfected HEK293 cells (l). HM form of WT-ABCG8 was quantified (m) (n = 6 for si-GL2, n = 3 for si-HRD1). (n) ABCG8 protein transfected in HEK293 cells was immunoprecipitated and immunoprecipitants were analyzed by Western blotting (IB). Input protein, cell lystes without immunoprecipitation of antibodies, was used as a positive control (input). Gels have been cropped for clarity; the bands were confirmed by the comparison with full-length gel images (Supplementary Figure S4) and molecular weight.