Extended Data Fig. 3: A photoactivatable affinity probe-based approach identifies PGK1 as the relevant cellular target of CBR-470-1.
From: A metabolite-derived protein modification integrates glycolysis with KEAP1–NRF2 signalling
a, Structure of CBR-470-PAP. b, Relative ARE-LUC luminance values from IMR32 cells transfected with pTI-ARE-LUC and then treated with the indicated doses of CBR-470-PAP for 24 h (n = 3). c, Silver staining and anti-biotin western blots of ammonium sulfate fractionated lysates from UV-irradiated IMR32 cells treated with 5 μM for 1 h with or without CBR-470-1 competition (250 μM) (n = 3). Shown on the right are initial proteomic target results from gel-band digestion and LC–MS/MS analysis. d, Anti-biotin western blots from in vitro crosslinking assays with recombinant PGK1 and EBP1 in the presence of the indicated doses of CBR-470-PAP (n = 2). e, Anti-biotin western blot analyses from an in vitro crosslinking assay with recombinant PGK1 in the presence of CBR-470-PAP (1 μM) and indicated concentration of soluble CBR-470-1 competitor (n = 2). f, Anti-biotin western blot analyses of cells treated with 5 μM CBR-470-PAP after transduction with anti-PGK1 and anti-EBP1 shRNA for 48 h. Depletion of PGK1 protein selectively reduces CBR-470-PAP-dependent labelling (n = 2). g, Dye-based thermal denaturation assay with recombinant PGK1 in the presence CBR-470-1 (20 μM) or vehicle alone (n = 3). Calculated melting temperature (Tm) values are listed. h, i, Dose-dependent thermal stability assay of recombinant PGK1 and GAPDH in the presence of increasing doses of CBR-470-1 near the Tm of both proteins (57 °C) (n = 5) (h) or room temperature (n = 3) (i). Western blot of sample supernatants after centrifugation (13,000 r.p.m.) detected total PGK1 and GAPDH protein, which were plotted in Prism (below). j, ARE-LUC reporter activity in HEK293T cells with transient shRNA knockdown of ENO1 (n = 3). Data are mean ± s.e.m. of biologically independent samples.
