Fig. 6: Glyoxal affects posttranslational modification and dynamics of tubulin.

a Scheme of detected CML modification sites for tubulin α-1B chain. b HUVEC were treated with GO (48 h) and α/β-tubulin and nuclei (DAPI) were stained. Representative immunofluorescence pictures (upper panel) and the percentage of cells with intensively stained tubulin filaments per high-power field (HPF) are shown (lower panel). Scale bar = 20 µm. n = 4. c After treatment of HUVEC with GO (1 mM, 48 h), nocodazole (noco) was added (10 µM, 2 min) and α/β-tubulin and nuclei were stained. Representative pictures and the percentage of cells with intensively stained tubulin filaments per HPF are shown. Scale bar = 20 µm. n = 5. d Purified porcine tubulin incubated with GO for 30 min on ice was analyzed in immunoblots. n = 5. e Purified porcine tubulin was pre-incubated with GO (1 mM, 30 min, on ice), or vehicle and polymerization induced by addition of cofactors at 37 °C was monitored in a fluorescence-based assay. Controls: taxol (3 µM) or nocodazole (20 µM) added to vehicle-treated tubulin at the start of polymerization. n = 3 (taxol, nocodazole), n = 6 (control (Ctrl), GO) mean ± SEM. Statistical significance was analyzed using one-way repeated measurement ANOVA corrected via Holm–Šidák method for areas under the curves. * p < 0.05 vs. control. f HUVEC were synchronized via a double thymidine block and treated with GO subsequent to releasing the block. Cells were analyzed in immunoblots n = 5. g HUVEC were treated with GO (48 h). The absolute level of CML modification at K58 on tubulin beta-4B chain (TUBB4B) was determined by PRM using the synthetic reference peptide _INVYYNEATGGK[CML]YVPR. The bar plot shows the fraction of CML-modified tubulin at K58 relative to total TUBB4B. Total TUBB4B levels were determined using the synthetic peptide _INVYYNEATGGK. n = 3. h HUVEC were treated with GO (24 h) and relative K58 acetylation on TUBB4B was quantified by label-free mass spectrometry using the endogenous peptide _INVYYNEATGGK[Ac]YVPR. n = 3. b–d, f–h: Bar graphs show mean + SEM. Statistical significance was analyzed using one-way or two-way repeated measurement ANOVA corrected via Holm–Šidák method. * p < 0.05 vs. control, # p < 0.05 vs. respective non-nocodazole-treated cells (c) or vs. respective non-GO-treated cells (f). Source data are provided as a Source Data file. Specific p values are listed in Supplementary Data 6. Related to Supplementary Fig. 7.