Fig. 6

Co-existing eIF2α O-GlcNAcylation and phosphorylation regulate HO-1 expression in opposite manner. A O-GlcNAz-modified proteins in HEK293T cells overexpressing FLAG-tagged WT or phospho-mut eIF2α (left panel) and parental BT-549 cells (right panel) were pulled down and subjected to immunoblot analysis (n = 2). Negative controls were parental HEK293T cells without transient overexpression and BT-549 cells without GlcNAz labeling. HEK293T and BT-549 cells were treated with GlcNAz (50 μM, 48 h) in an -Arg medium prior to cell harvest. O-GlcNAz-modified proteins were collected and analyzed using an anti-FLAG or anti-eIF2α antibody to determine the FLAG-tagged exogenous or endogenous eIF2α O-GlcNAcylation levels. Black arrowheads indicate endogenous eIF2α, while white arrowheads indicate FLAG-tagged eIF2α. The asterisk (*) indicates phosphorylation of eIF2α on O-GlcNAz-modified eIF2α. B qRT-PCR analysis of HMOX1 mRNA levels in parental BT-549 and eIF2α (WT or mutants) overexpressing cells subjected to ArgS for 24 h; n = 4. On the left, cells were transfected with FLAG-tagged WT or phospho-mimicking (S51D) eIF2α (2 μg) for 24 h prior to ArgS. Parental BT-549 cells serve as the baseline for HMOX1 expression. On the right, BT-549 cells stably overexpressing FLAG-tagged WT, phospho-mut, O-GlcN-mut, and quadruple-mut eIF2α were transfected with siRNA for eIF2α (30 nM) for 48 h prior to ArgS. HMOX1 mRNA levels were determined by qRT-PCR and analyzed as described in Fig. 5C. Data are shown as mean ± s.e.m.; *p < 0.05; ***p < 0.001; Two-Way ANOVA followed by Tukey's multiple comparison test