Fig 1: Activation of the eIF2α-ATF4 signalling pathway enhances the flux of the HBP upon glucose deprivation.(a) HBEC 3KT-RL cells incubated with 0.1 mM Glc over a time course of 24 hours were analyzed by Western blot developed with antibody against O-GlcNAc-modified proteins. Quantification of the intensity of O-GlcNAcylated protein bands of independent experiments, compared to 0 h condition and normalized to the loading control tubulin is shown. (b) Western blot analysis of O-GlcNAcylated proteins and ATF4 protein abundance in cells transfected with a control siRNA (siCtrl) or an ATF4 siRNA and incubated with 25 mM Glc (+) or 0.1 mM Glc (−) for 24 hours. Samples derive from the same experiment and gels/blots were processed in parallel. Quantification of the intensity of O-GlcNAcylated protein bands of of independent experiments and normalized to the loading control tubulin is shown. (c) Rat1 cells stably expressing the Fv2E-PERK construct and treated with 2 nM AP20187 over a time course were analyzed by Western blot developed with antibody against O-GlcNAc-modified proteins. Arrowheads exemplify bands with the substantial changes in O-GlcNAcylation levels. Quantification of the intensity of O-GlcNAcylated protein bands of of independent experiments, compared to 0 h condition and normalized to the loading control tubulin is shown. Full-length blots/gels are presented in Supplementary Figure S7. **p < 0.01, ***p < 0.001.
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