Supersignal west pico chemiluminescent system

Samples were loaded on SDS-PAGE. Proteins were then transferred onto nitrocellulose membranes and blocked with 5% powdered nonfat milk in PBS-T (Tween 0.1%). Primary antibody was incubated overnight at 4 °C in 5% powdered nonfat milk in PBS-T (Tween 0.1%). After three washes in 5% powdered nonfat milk in PBS-T, the secondary HRP-coupled antibodies were incubated for 1 h at room temperature in the same buffer. The signal was detected using the Super Signal West Pico chemiluminescent system (Thermo Fisher Scientific).

Cells were stimulated for 30 min or 5 h with butyrate, propionate, or TSA, washed and lysed directly in Laemmli sample buffer (62 mM Tris/HCl pH 6.8; 2% SDS; 10% glycerol; 5% 2-mercaptoethanol; 0.01% bromophenol blue). Cell lysates were briefly sonicated, separated on 15% SDS-polyacrylamide gels and transferred to Immobilon-P polyvinylidene difluoride (PVDF) transfer membranes (0.45 μm, Millipore, Amsterdam, The Netherlands). Blots were blocked with PBS/2% milk/0.1% Tween20 and immunostained o/n at 4^°C with a polyclonal rabbit antibody against acetylated-lysines (#9441, Cell Signaling Technology, Bioké, Leiden, The Netherlands) at a 1:1,000 dilution. Other polyclonal antibodies used were directed against acetyl-H3K9 (Merck, 07-352), acetyl-H4K5 (Merck, 07-327), acetyl-H4K16 (Merck, 07-329), HDAC1 (Abcam, ab109411), HDAC2 (Abcam, ab124974) and HDAC3 (Abcam, ab16047). As a secondary antibody, HRP-conjugated goat anti-rabbit antibody (Santa Cruz Biotechnology, Heidelberg, Germany) was used. As a loading control, GAPDH antibody (Sigma-Aldrich) was used at 1:10,000 dilution. Signal was visualized with the SuperSignal West Pico Chemiluminescent system (Thermo Scientific, Rockford, IL, USA) in a ChemiDoc MP (Biorad).

Cells were seeded at a density of 5×10⁵ cells/well in 6-well plates. Twenty-four hours post seeding, media was removed, cells were rinsed with PBS, and serum-free media containing Dacomitinib or DMSO was added to each well. Twenty-four hours post treatment, the EGFR ligand epidermal growth factor (EGF; Sigma), was added to one set of the treatment plates to a final concentration 20 ng/mL, then incubated for an additional 30 minutes. After EGF treatment, media from all plates was removed; cells were washed with PBS, and lysed at 4°C using a lysis buffer containing protease and phosphatase inhibitors (Roche). Twenty micrograms of extracted protein for each sample was separated on a 4–20% gradient polyacrylamide Tris-glycine gel (Life Technologies; Foster City, California) and transferred to PVDF membranes (Millipore). Membranes were incubated overnight at 4°C with specific antibodies against EGFR (#4267), phospho-EGFR (#3777), ERK (#4695), phospho-ERK (#4370), AKT (#4691), phospho-AKT (#4060), mTOR (#2983), phospho-mTOR (#2971), STAT3 (#9139), and phospho-STAT3 (#9145; all from Cell Signalling, 1∶1000 dilution). Blots were then incubated with horseradish peroxidase conjugated to anti-rabbit or anti-mouse antibody (1∶5000; Biorad), then analyzed with Supersignal West Pico Chemiluminescent system (Thermo Scientific).

Epimastigotes, in vitro-derived metacyclic trypomastigotes and isolated intracellular amastigotes obtained as described above (see “Parasites”) were washed in PBS, resuspended in PBS containing a protease inhibitor cocktail (Roche, Basel, Switzerland), and lysed by the addition of 4x Laemmli sample buffer, which was followed by heating at 100°C for 5 min. Lysates containing an equivalent of 1 x 10⁶ cells μl^-1 were fractionated by SDS-PAGE and transferred onto nitrocellulose membranes (Hybond C, GE Healthcare, PA, USA) according to standard protocols [39 ]. Membranes were blocked with 5% non-fat dry milk and 0.05% Tween 20 in PBS (blocking solution) then, they were probed with one of the following primary antibodies (diluted in blocking solution): the monoclonal antibody 211.F7, against the Tcγ hinge domain (1:300); anti-cruzipain (1:1500) antiserum that recognizes both the zymogen and the mature form of cruzipain [40 ]; or a mouse anti-TcGAPDH antiserum (1:8,000) [41 ], as a loading control. Membranes were then incubated with horseradish peroxidase-conjugated anti-mouse IgG antibodies (Thermo Scientific product #31430) and diluted to 1:7,500 in blocking solution. The SuperSignal West Pico Chemiluminescent System (Thermo Scientific) was used for antibody binding detection according to the manufacturer’s instructions.