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. 2022 Aug 3;298(9):102340. doi: 10.1016/j.jbc.2022.102340

Figure 2.

Figure 2

EGF increases PKM2 association with OGT.A, EGF increases the interaction between PKM2 and OGT. (top) A549 cells treated by EGF (100 ng/ml for 1 h) were prepared for IP with PKM2 or OGT antibody and analyzed by WB with indicated antibodies. (bottom) Relative protein levels of IP. OGT, OGA, and PKM2 O-GlcNAc were normalized to immunoprecipitated PKM2, while OGT pTyr and PKM2 were normalized to OGT. Quantification shows mean ± SD (n = 3) with significance determined by student’s t test, ∗∗∗p < 0.001, ns, nonsignificant. B, activated EGFR increases PKM2 binding to OGT. (top) In A549 cells deprived for serum and transfected with EGFR or EGFRvIII for 24 h, PKM2- or OGT-associated proteins were obtained from IP and examined by WB with indicated antibodies. (bottom) Relative protein levels of IP. OGT, OGA, PKM2 O-GlcNAc, OGT pTyr, and PKM2 were normalized to PKM2 or OGT. Quantification shows mean ± SD (n = 3) with significance determined by one-way ANOVA, ∗∗∗p < 0.001, ns, nonsignificant. C, assay for UDP-GlcNAc level. A549 cells were treated with EGF (100 ng/ml) for different durations and subjected to UDP-GlcNAc analysis by HPLC. Data represent mean ± SD (n = 3) with significance determined by one-way ANOVA, ns, nonsignificant. EGF, epidermal growth factor; EGFR, EGF receptor; OGA, O-GlcNAcase; OGT, O-GlcNAc transferase.