Nupage 4 12 gel

Western blotting (WB) procedures were essentially conducted as previously described [7 (link)]. Briefly, SDS-PAGE (NuPAGE 4–12% gels, Life Technologies) was used to separate total cellular proteins from Jurkat T cells (ATCC^® TIB-152™) or prostate cancer PC3 cells (ATCC^® CRL-1435™), followed by transfer to nitrocellulose membranes (GE Healthcare Life Sciences). Membranes were cut into individual strips and blocked overnight with 5% dry milk solution in TBS-T buffer (20 mM Tris–HCl, pH 7.6, 140 mM NaCl, 0.1% Tween 20) and individual strips were then probed with different patient sera for 2 h. After several washes with TBS-T, the individual membrane strips were incubated for 1 h with horseradish peroxidase (HRP)-conjugated goat anti-human secondary antibody (ThermoFisher Scientific, Cat# A18847, used at 1:5000 in 5% milk/TBS-T). Detection of serum autoantibodies bound to proteins was achieved by enhanced chemiluminescence (ECL) (Thermo Fisher Scientific Pierce) in autoradiography films.

HepG2 cells were acquired from European Collection of Cell Cultures (ECACC, Salisbury, UK). HepaRG cells, basal media, growth and differentiation additives were purchased from Biopredic International (Saint Grégoire, France). Dulbecco's modified media (DMEM) high glucose, fetal bovine serum (FBS), Cell Tracker 5- chloromethylfluorescein diacetate (CMFDA), NUPAGE 4–12% gels, rat tail collagen I and phosphate buffered saline (PBS) were purchased from Life Technologies (Paisley, UK). Nitrocellulose membrane and enhanced chemiluminescence (ECL) were purchased from GE Healthcare (Buckinghamshire, UK). All Seahorse consumables were purchased from Agilent (Santa Clara, USA). High precision pump – pump 11 was purchased from Harvard apparatus (Massachusetts, USA). 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl benzimidazol carbocyanine iodide (JC-1) was purchased from Abcam (Cambridge, UK). Cytotoxicity Detection Kit was purchased from Roche Diagnostics Ltd. (West Sussex, UK). Balch homogeniser was purchased from Isobiotech (Heidelberg, Germany). Williams' E Medium powder (with l-Glutamine, without glucose) was manufactured by United States Biological. All bile acids, bile salts, MK571, bosentan were purchased from Sigma Aldrich (Dorset UK).

For glutathione S-transferase (GST)-spastin pull-down assays, a liquid chromatography-mass spectrometry (LC-MS) approach was employed to detect precipitated proteins. Briefly, proteins from samples of T10-centered spinal cord tissue were resolved using NuPAGE 4–12% gels (Life Technologies), visualized with a Colloidal Blue Staining Kit (Life Technologies), and target protein bands were then excised, digested using trypsin, and the resultant peptides were assessed via nanoflow reversed-phase liquid chromatography-tandem MS utilizing an HPLC Ultimate 5600 system (AB SCIEX, CA, United States) with a linear ion trap (ThermoElectron, MA, United States) in data-dependent acquisition mode.

Recombinant GFP-HIPK2, GFP-K565T and GFP-T566P were produced in U2OS cells by transfection with Lipofectamine (Invitrogen). Total cell extracts were prepared 24 hrs post-transfection by incubation for 30 min at 4°C in lysis buffer [50 mM Tris-HCl (pH 7.4), 300 mM NaCl, 1% Nonidet P-40, 5mM EDTA, protease and phosphatase inhibitor (Roche)]. When required, to reduce protein phosphorylation, the total cell extracts were incubated at 30°C for 30 min in the presence of 20 U/ml calf intestinal alkaline phosphatase (Sigma). After centrifugation, GFP-fusion proteins were purified from supernatant by overnight incubation with anti-GFP Ab-sepharose beads (Abcam) at 4°C and used as enzymatic source. Western blot (WB) analysis with a different anti-GFP Ab (mouse monoclonal anti-GFP Ab, Roche) was employed to quantify purified proteins. For kinase assay, recombinant proteins were incubated with recombinant p53, p63, and MBP for 30 min at 30°C in a kinase buffer in the presence of [γ³²P]-ATP, as described [15 (link)]. The phosphorylated substrates were resolved on precast NuPAGE 4-12% gels (Thermo Fisher Scientific) and analyzed by autoradiography.

Glycan composition analysis was performed by Proteodynamics (Riom, France). N-linked glycans were removed from HEK 293T derived GP_1,2 using 15 U per 200 µg protein of PNGase F (Promega, Madison, WI, USA) for 15 hours at 37 °C in phosphate buffer (pH of 7.5) while Sf9 cell derived protein was deglycosylated with 15 U of PNGase F (Promega, Madison, WI, USA) and 10 µL of PNGAse A (Sigma) for 15 hours at 37 °C in 50 mM sodium acetate buffer, pH 5.0. To confirm deglycosylation, electrophoresis on NuPAGE 4–12% gels (ThermoFisher Scientific, Waltham, MA) was performed. The N-glycans were then purified and permethylated according to Morelle et al.^{43 (link)}.