FIGURE 5.

N-terminal cleavage and O-glycosylation of hβ₁AR in glycoengineered HepG2 cell lines. The WT Myc- and FLAG-tagged hβ₁AR and the N-terminally truncated (Δ2–31 and Δ2–52) mutants with a C-terminal FLAG tag were transiently expressed in the indicated cell lines for 48 h. Receptors were immunoprecipitated from cellular lysates and analyzed by SDS-PAGE and Western blotting. In A, a shorter exposure is shown for the outlined area of lane 2. The ratios of the WT full-length (black bars) and cleaved receptor forms (white bars) shown in A are depicted in B. Two-way analysis of variance followed by Tukey's post hoc test was used to compare the ratios in HepG2ΔT2 and HepG2ΔT2+T2 cells with the corresponding values in the HepG2WT cells (a). The values obtained for the ΔT2 and ΔT2+T2 cells were also compared (b). ***, p < 0.001; ns, not significant; n = 3–5. For D, immunoprecipitated receptors were subjected or not to deglycosylation with neuraminidase and O-glycosidase before SDS-PAGE. The results shown are representative of five (A, lanes 1–3), three (A, lanes 4–6), four (C), and one (D) independent experiment. Open circles, full-length hβ₁ARs; open triangles, C-terminal fragments cleaved at ³¹R↓L³². IP, immunoprecipitation; WB, Western blotting. Error bars, S.E.