Skip to main content
. 2022 Aug 3;298(9):102341. doi: 10.1016/j.jbc.2022.102341

Figure 2.

Figure 2

Upon elevated O-GlcNAcylation, ULK1 is O-GlcNAcylated at Ser409.A, HeLa cells were transfected with HA-OGT and FLAG-ULK1 plasmids and then treated with 30 mM TMG plus glucose (TMG + Glu) or left untreated. Cells were then subjected to immunoprecipitation and immunoblotting with indicated antibodies. B, HeLa cells were treated with TMG + Glu or left untreated, immunoprecipitated with anti-ULK1 antibodies, and immunoprecipitated with anti-ULK1 or RL2 antibodies. C, HeLa cells were transfected with FLAG-ULK1 plasmids and enriched for O-GlcNAc by TMG + Glu treatment. Cell lysates were immunoprecipitated with anti-FLAG M2 agarose and then subjected to silver staining. The asterisk denotes FLAG-ULK1. D, ETD MS/MS spectrum of the O-GlcNAcylated peptide TPSPSPPC(carbamidomethyl)S(HexNAc)SSPSPSGR on ULK1. The matched fragment ions are shown in red and blue. E, HeLa cells were transfected with HA-OGT together with ULK1-S409AS410A (2A). Cells were then treated with TMG + Glu, and the resultant lysates were collected and subjected to immunoprecipitation and immunoblotting. ∗∗∗ indicates significant differences as determined by one-way ANOVA (p < 0.001). ETD, electron transfer dissociation; HA, hemagglutinin; O-GlcNAc, O-linked β-N-acetylglucosamine; OGT, O-GlcNAc transferase; TMG, Thiamet-G; ULK1, Unc-51-like kinase 1.