In both, proteins related to cell redox homeostasis are highlighted in red. ASH2L and CBX3 proteins from LG-ITRR experiment. C, Western blot validation of thermal stabilization of PRMT1, ASH2L and CBX3 proteins. D, Structure of human HAT1 (PDB accession 2P0W) (gray) showing the position of predicted redox-sensitive cysteines C27 (yellow spheres), C101 (blue spheres) and C168 (orange spheres). E, Distinct thermal stability changes of histone variants in the LG-ITRR experiment. Yellow and green curves (left) represent the ITRR response of cysteine-bearing histone H3 variant while gray and brown curves for the ITRR response of non-cysteine containing histone H1 variants. mmc1.pdf (4.1M) GUID:?B2D271A1-9B7B-4276-8329-94208E03AF8A Supplementary Fig. 2 CETSA shifts related to protein complex formation and metabolite concentration changes upon H2O2 exposure. Related to Fig. 3. A, Decreased GSH/GSSG ratios and GSH amounts upon H2O2 treatment in Rabbit Polyclonal to GPR158 HepG2 cells. Statistical significance was calculated with Aldoxorubicin two sample (Novagen) in Terrific Broth media supplemented with kanamycin and chloramphenicol. Cells were cultured and induced with 0.5?mM isopropyl-beta-d-1-thiogalactopyranoside (IPTG) at 18?C overnight, harvested and resuspended in lysis buffer (100?mM HEPES, 500?mM NaCl, 10?mM imidazole, 10% (v/v) glycerol at pH 8.0) supplemented with 1:1000 (v/v) EDTA-free protease inhibitor cocktail (Nacalai) and 250U/ml of Benzonase (Merck). After sonication, centrifugation and clarified by filtration, the protein extract was loaded onto Ni-NTA Superflow column (Qiagen), washed and eluted with 5 column volumes of elution buffer (20?mM HEPES, 500?mM NaCl, 500?mM imidazole, 10% (v/v) glycerol at pH 7.5). Eluate was then loaded onto a HiLoad 16/60 Superdex-200 column (GE Healthcare) and eluted with Aldoxorubicin 1 column volumes of elution buffer (20?mM HEPES, 300?mM NaCl, 10% (v/v) glycerol at pH 7.5). Relevant protein fractions were pooled and concentrated using centrifugal force driven filter concentrators (VivaScience). Protein purity was assessed on SDS-PAGE and identity confirmed by mass spectrometry analysis. Protein concentration was determined by the absorbance at 280?nm using Nanodrop spectrophotometer (ThermoFisher Scientific). 2.8. In vitro reduction and oxidation HepG2 cell extracts and purified HAT1 recombinant proteins, which were prepared as mentioned were treated by 1?mM GSSG alone or 1?mM GSSG in combination with 5?mM GSH for 10?min at room temperature. Free glutathione was Aldoxorubicin removed by diluting the reaction with 10?vol designated buffers and filtering through Vivaspin 500 concentrator (Sartorius). Samples were then supplemented with NuPAGE LDS sample buffer (ThermoFisher Scientific) in the absence of reducing agent and without boiling; or in the presence of 100?mM DTT Aldoxorubicin and boiled at 95?C for 10?min, and used for western blotting analysis. 2.9. Western blot analysis 20?g of total proteins from cell lysate or 200?ng of recombinant proteins were separated on NuPAGE Bis-Tris 4C12% Protein Gels (Invitrogen) and transferred to nitrocellulose membranes using iBlot system (Invitrogen). Membrane was blocked by 5% skimmed milk and incubated with primary antibodies for designated protein detection. The following antibodies were used in this study: anti-PRDX1 (#8499), anti-CBX3 (#2619) and anti-UBA2 (#8688) antibodies from Cell Signaling Technology; anti-AHS2 (A300-489A) from Bethyl Laboratories; anti-HAT1 (sc-390562) and anti-PRMT1 (sc-166963) from Santa Cruz Biotechnology. Membrane was then washed with PBS containing 0.1% Tween 20 (Sigma Aldrich) and incubated with corresponding secondary antibodies accordingly. Goat anti-mouse (#31430) or anti-rabbit (#31460) IgG (H+L) secondary antibodies were obtained from ThermoFisher Scientific. After thorough washing of membranes, chemiluminescence signals were visualized using Clarity ECL blotting substrates (Bio-Rad) and captured by ChemiDoc MP imaging system (Bio-Rad). 2.10. Protein interaction network generation and gene ontology analysis Protein interaction network among CETSA hits of each treatment was retrieved by importing a list of Uniprot IDs into Cytoscape v.3.7.0 (https://cytoscape.org). In the embedded STRING interaction database (http://apps.cytoscape.org/apps/stringApp), a default confidence score cut-off at 0.4 was applied for each network Aldoxorubicin retrieval. Each node represented one hit protein and edge width represented interaction score. Thermal shift profiles of each hit were mapped with a numeric table of corresponding thermal shift ratio and visualized.