Fig. 4

eIF2α O-GlcNAcylation suppresses HO-1 protein expression in Arg-starved breast cancer cells. A Immunoblot analysis of HO-1 in BT-549 cells subjected to ArgS for 24 h. One representative immunoblot is shown in the left panel (n = 4). BT-549 cells were transiently transfected with FLAG-tagged WT, O-GlcN-mut, or phospho-mut eIF2α. Parental BT-549 cells labeled with (-) serve as a negative control of eIF2α overexpression. The relative levels of HO-1 (middle panel) and p-eIF2α (right panel) are expressed as fold change and used to compare protein levels across experimental conditions. B Immunoblot analysis of HO-1 in BT-549 cells stably WT, phospho-mut, O-GlcN-mut, or quadruple-mutant eIF2α subjected to ArgS for 24 h. One representative immunoblot is shown in the left panel (n = 4). Parental BT-549 cells labeled with (-) serve as a negative control of eIF2α overexpression. The relative levels of HO-1 protein (middle panel) and p-eIF2α (right panel) are expressed as fold change and used to compare protein levels across experimental conditions. A, B Black arrowheads indicate endogenous eIF2α, while white arrowheads indicate FLAG-tagged eIF2α. The relative p-eIF2α level is calculated by normalizing the densitometric tracing of the p-eIF2α signal with the total eIF2α signal. The ratio in each experimental condition is then compared to the reference (parental; + Arg; set as 1). The relative HO-1 protein level is determined by comparing the densitometric tracing of HO-1 signal in experimental conditions to the reference, with the values of the reference HO-1 and p-eIF2α set as 1 after normalization with H3 (used as a loading control). Data are presented as mean ± standard error of the mean (s.e.m.); *: p < 0.05; **: p < 0.01; ***: p < 0.001. Statistical analysis was performed using Two-Way ANOVA followed by Dunnett's multiple comparison test