Isopropyl 1 thio β d galactopyranoside

Uridine 5′-diphospho-N-acetylglucosamine sodium salt (U4375) and isopropyl 1-thio-β-D-galactopyranoside was purchased from sigma Aldrich (Zwijn-drecht, The Netherlands). The mouse monoclonal anti-Anti-O-Linked N-Acetylglucosamine antibody [RL2] (ab2739) was from Abcam (London, England). FITC conjugated secondary antibody was purchased from Thermo scientific (Bleiswijk, Netherland). A known OGT inhibitor 3-(2-adamantanylethyl)-2-[(4-chlorophenyl) azamethylene]-4-oxo-1,3-thiazaperhyd roine-6-carboxylic acid was obtained from TimTec (Newark, USA). All PamChip 4 microarray chips were provided by PamGene (Pamgene international, The Netherlands).

Tris and diethypyrocarbonate were purchased from Amresco (Solon, OH, USA). Mouse anti-Flag-tag monoclonal antibody, anti-Flag M2-agrarose, protease inhibitor cocktail, leupeptin, phenylmethylsulfonyl fluoride (PMSF), Triton X-100, Tween-20, Nonidet P40 (NP-40), hydroxyl urea (HU), ribonuclease A (RNase A), bovine serum albumin (BSA), paraformaldehyde, imidazole, sodium dodecyl sulfate (SDS), guanidine hydrochloride (GdnHCl), polyadenylic acid potassium salts (catalog number: P9403-25MG), DTT, kanamycin, ampicillin and isopropyl-1-thio-β-D-galactopyranoside (IPTG) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Rabbit anti-PARN and mouse anti-nucleophosmin (NPM1) antibodies were from Abcam (Cambridge, MA, USA). FITC AffiniPure goat anti-mouse IgG, FITC AffiniPure goat anti-rabbit IgG, mouse anti-HA, rabbit anti-HA, DyLight 594 AffiniPure goat anti-mouse, DyLight 594 AffiniPure goat anti-rabbit, HRP AffiniPure goat anti-mouse and HRP AffiniPure goat anti-rabbit antibodies were from EarthOx (Millbrae, CA, USA). The Annexin V-FITC/PI apoptosis detection Kit was obtained from Bioworld (Louis Park, MN, USA). The transfection reagent VigoFect was from Vigorous (Beijing, China). Hoechst 33342 was from Invitrogen (Carlsbad, CA, USA). All other reagents were local products of analytical grade.

1-Anilino-8-naphthalene sulfonic acid (ANS), Thioflavin T (ThT), α-chymotrypsin, kanamycin, isopropyl-1-thio-β-d-galactopyranoside (IPTG), α-glucosidase (α-Gls), dithiothreitol (DTT), 2,5-diphenyltetrazolium bromide (MTT) and bovine pancreatic insulin were purchased from Sigma. Additionally, we used ethylenediaminetetraacetic acid (EDTA), urea, β-mercaptoethanol (β-ME), and various other chemicals, which were provided by Merck.

The synthetic gene encoding for bovine S100B was cloned into pAED4 plasmid and expressed in Escherichia coli utilizing the T7, AmpR expression system. Bacterial cells (HMS174 (DE3)) were grown in LB medium at 37 °C. Expression was induced by the addition of 0.4 mM isopropyl 1-thio-β-d-galactopyranoside (Sigma-Aldrich) at A600 = 0.8, and the bacterial culture was raised for two hours. The overexpressed protein was purified as described previously [49 (link)]. Analytical reverse phase HPLC and mass spectrometry confirmed the absence of any fragmented or modified protein. Protein concentration was estimated from its absorbance at 280 nm. The molar extinction coefficient for S100B protein (ϵmolar = 10.026) was determined experimentally by measuring the UV signal at 280 nm for a standard S100B solution. The standard S100B sample concentration was established by amino acid analysis at BioCentrum Ltd. (Kraków, Poland). Namely, the protein samples were hydrolyzed in the gas phase using 6 M HCl at 115 °C for 24 h. The free amino acids were converted into phenylthiocarbamyl derivatives and analyzed by high-pressure liquid chromatography (HPLC) on a PicoTag 3.9 × 150-mm column (Waters, Milford, MA, USA).