Figure 2
Co- and post-transcriptional N-glycosylation in human ABCG5 and ABCG8.
(a), (b) STT3 siRNAs (100 nM)-transfected HEK293 cells were co-transfected with Myc-ABCG5 (a) or HA-ABCG8 (b). Cell lysates were analyzed by immunoblotting. (c) Knockdown efficiencies of STT3A and STT3B genes were analyzed by quantitative RT-PCR (Q-RT-PCR). Human 18s ribosmal RNA (18srRNA) was used as an internal control. *p < 0.05, ***p < 0.001, versus con siRNA-transfected cells; Dunnett's test (n = 3). (d), (e) Myc-ABCG5 or HA-ABCG8-transfected HEK293 cells were pulse labeled for indicated time (5, 15, 30 min) and cell lysates were harvested immediately and digested with or without EndoH. Samples were immunoprecipitated with anti-Myc or anti-HA antibody, followed by autoradiography (d). Relative quantity of each glycosylated form of ABCG5 or ABCG8 within all ABCG5 or ABCG8 bands in a short pulse-labeling period (5 min) was quantified (e). (f–i) Pulse chase (pulse labeled for 10 min) of HEK293 cells expressing Myc-tagged WT-, N584Q- and N591Q-ABCG5 (f). Relative quantity of each glycosylated form of WT-ABCG5 (g), N591Q-ABCG5 (h) and N584Q-ABCG5 (i) within all ABCG5 was quantified. (j), (k) Pulse chase of HEK293 cells expressing HA-tagged WT-ABCG8 (j) and relative quantity of each glycosylated form of WT-ABCG8 within all ABCG8 (k). Gels have been cropped for clarity; the bands were confirmed by the comparison with full-length gel images (Supplementary Figure S4) and molecular weight.
