Proteinase inhibitor cocktail mix

Total protein was extracted from the cells in a lysis buffer containing 1% NP-40 (Fluka), 20 mM Tris–HCl pH 7.4 (Tris MP Biomedicals, HCl Sigma), 140 mM NaCl (VWR), and 2 mM EDTA (MP Biomedicals); supplemented with 100 mM orthovanadate (Sigma), 200 mM PMSF (Sigma), proteinase inhibitor cocktail mix (Roche), phosSTOP (Roche), and 0.1 mM DTT (Invitrogen). Protein fractions were separated by SDS-PAGE and electrotransferred onto nitrocellulose membranes (Biorad). The following primary Abs were used: mouse anti-phosphorylated Y701 STAT1 (BD), mouse anti-STAT1 (BD), rabbit anti-phosphorylated Y705 STAT3 (Cell Signaling Technology), rabbit anti-STAT3 (Cell Signaling Technology), goat anti-IL-10RB (Bio-Techne), mouse HRP-conjugated anti-His (Santa Cruz Biotechnology), mouse anti-V5 (Invitrogen), mouse anti-GAPDH (Santa Cruz Biotechnology) or rabbit anti-GAPDH (Santa Cruz Biotechnology), and mouse HRP-conjugated anti-DDK (Origene). Antibody binding was detected by incubation with HRP-conjugated anti-mouse, anti-goat, or anti-rabbit secondary Abs (GE Healthcare), with the ECL system (Thermo Fisher Scientific). Antibodies were also detected by fluorescence on the Licor system with secondary antibodies conjugated to IRDye800 or IRDye680 anti-mouse, anti-goat, or anti-rabbit antibodies (all from Licor Biosciences Proteomics).

Cells were lysed using a modified version of the RIPA buffer (50 mM Tris-HCl pH8, 0.5% NaDeoxycholate, 150 mM NaCl, 1% NP-40, 0.1% SDS) supplemented with proteinase inhibitor cocktail mix (Roche, 11 873 580 001), sonicated (Bioruptor, 10 cycles, 10 seconds, high energy), and centrifuged for 15 minutes at 4°C. The cell lysates (supernatants) were quantified using Bradford reagent (Bio-rad, 500–0205), separated on the Bio-rad Minigel system, and blotted onto nitrocellulose membranes (Bio-rad, 162–0115), which were blocked using StartingBlock T20 blocking buffer from Thermo Scientific (37543). Antibodies used were listed in S6 Table. Signals were detected using the WesternBright ECL detection kit from Advansta (K-12045-D20).

Total proteins were solubilized in RIPA buffer (Sigma, Saint Quentin Fallavier, France) supplemented with 1× proteinase inhibitor cocktail mix (Roche, Sigma). Following separation by SDS–PAGE, immunoblotting was performed using Ab against ALPI (ab54776, 1 μg/ml, Abcam, Paris, France) and GAPDH (3683, 1:2,000, Cell Signaling). Surface cell staining of transduced cell lines was performed according to standard flow cytometry protocol using primary Ab against ALPI (ab54776, 1 μg/ml, Abcam), and secondary BV510 goat anti‐mouse (Poly4053, 1:100, Sony Biotechnologies, Weybridge, UK) was used. Data were analysed on FACS Canto II (BD Biosciences, Rungis, France) using FlowJo software.