Bis tris mini gel

LC–MS/MS analysis of L. arvalis, A. elegans, T. ljubarskyi and T. reesei secretomes was performed as described [26 (link)]. Briefly, short SDS-PAGE runs (pre-casted Bis–Tris Mini Gels, Invitrogen, France) were performed, allowing 10 µg of proteins diafiltered from secretomes to migrate on 0.5 cm length. Each one-dimensional electrophoresis lane was cut into two slices of gel and protein identification was performed using PAPPSO “Plate-forme d’Analyse Protéomique de Paris Sud-Ouest” platform facilities. In-gel digestion was carried out according to a standard trypsinolysis protocol. Online analysis of peptides was performed with a Q-exactive mass spectrometer (Thermo Fisher Scientific, USA), using a nanoelectrospray ion source. Protein identification was performed by querying MS/MS data against the corresponding genome available at the Joint Genome Institute [32 (link)] for A. elegans, T. ljubarskyi and T. reesei strains, and against the transcriptome of L. arvalis (BioProject Accession: PRJNA244907), together with an in-house contaminant database, using the X!Tandem software (X!Tandem Cyclone, Jouy en Josas, France). All peptides matched with an E value lower than 0.05 were parsed with X!Tandem pipeline software. Proteins identified with at least two unique peptides and a log (E value) lower than − 2.6 were validated.

LC–MS/MS analysis of P. coccineus secretomes, PcoAspen, PcoPine and PcoMaltose, was performed as described in Ref. [50]. Briefly, short SDS-PAGE runs (pre-casted Bis–Tris Mini Gels, Invitrogen, France) were performed, allowing 10 µg of proteins diafiltered from secretomes to migrate on 0.5 cm length. Each one-dimensional electrophoresis lane was cut into two slices of gel and protein identification was performed using PAPPSO “Plate-forme d’Analyse Protéomique de Paris Sud-Ouest” platform facilities. In-gel digestion was carried out according to a standard trypsinolysis protocol. Online analysis of peptides was performed with a Q-exactive mass spectrometer (Thermo Fisher Scientific, USA), using a nanoelectrospray ion source. Protein identification was performed by querying MS/MS data against the Pycnoporus coccineus genome (http://genome.jgi.doe.org/Pycco1/Pycco1.home.html), together with an in-house contaminant database, using the X!Tandem software (X!Tandem Cyclone, Jouy en Josas, France). All peptides matched with an E value lower than 0.05 were parsed with X!Tandem pipeline software. Proteins identified with at least two unique peptides and a log (E value) lower than −2.6 were validated.

Proteins extracted from mice tumor were resolved by SDS/PAGE on precast 4–12% Bis‐Tris minigels (Life Technologies). Following staining with NuPage Colloidal Coomassie (Life Technologies), gel lanes were cut into 10–12 slices, and in‐gel tryptic digestion and liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) were performed as already described (Boussadia et al., 2018). Data acquisition was performed in data‐dependent Top5. Spectra files were analyzed by Sequest HT search engine with proteome discoverer 1.4 (Thermo Fisher) using the Human Uniprot‐Swissprot database (released on June 2016) and containing also decoy database. The carbamidomethylation of cysteines was specified as a fixed modification, and the oxidation of methionine and phosphorylation of serine, threonine and tyrosine were set as variable modifications; mass tolerance was set to 1 Da for precursor ion and 0.4 Da for fragment ions; a maximum of two missed cleavages was allowed. The Percolator tool was used for peptide validation based on the q‐value and high confidence was chosen, corresponding to a false discovery rate ≤ 1% at peptide level. Proteins were identified with a minimum of one peptide rank = 1. Protein abundance was determined according to the label‐free Top3 method as already described (Fratini et al., 2017).

100 ng of recombinant human IDH1 R132H protein and wild type IDH1 protein (Abcam, Cambridge, MA, USA) were loaded onto 4–12% Bis-Tris mini gels (Life Technologies, Carlsbad, CA, USA) and blotted to PVDF membranes (iBlot™ Transfer Stack, PVDF, Invitrogen, Carlsbad, CA, USA). The Western blot was carried out using WesternBreeze Chromogenic kit (Invitrogen, Carlsbad, CA, USA) and the supernatant of the transfected cells (0.5 µg/mL IgG) or H09 (0.5 µg/mL antibody) were used to probe the membranes.

Western blotting was performed to confirm the expression of HSP110 and β-actin in gastric cancer cell lines. Transfected cells were incubated for 72 h, and total proteins were extracted from MKN7, MKN45, MKN74, and AZ521 cells using PRO-PREP Protein Extraction Solution Kit (iNtRON Biotechnology, Sungnam, Kyungki-Do, Korea). The proteins were separated on 4–12% Bis-Tris Mini Gels (Life Technologies Corporation, Carlsbad, CA, USA), and transferred to membranes using an iBlot Dry Blotting System (Life Technologies Corporation, Carlsbad, CA, USA). The membranes were incubated overnight at 4°C with rabbit monoclonal anti-HSP110 antibody (1:1000; GeneTex, CA, USA) and anti-β-actin antibody (1:1000; Sigma-Aldrich, St Louis, MO, USA). Following this, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibodies, and the target proteins were detected with the ECL Prime Western Blotting Detection System (GE Healthcare, Tokyo, Japan) using Image Quant LAS4000 (GE Healthcare Life Sciences, UK).