Chemidoc station

C2C12 myoblasts (2000 cells/well) were seeded in 96-well black plates. After 24 h, cells were treated for 24 h with compounds of interest. Caspase3/7 activation was determined using the Caspase-Glo 3/7 Assay System (cat# G8090, Promega). Luminescence was measured with a Promega GloMax Plate Reader. Chemiluminescent signals were acquired using a ChemiDoc station (Biorad, Segrate, Italy).

BMDCs were washed and resuspended at 6 × 10⁶cells/mL in RPMI‐1640 with L‐glutamine supplemented with β‐mercaptoethanol (50 μM), penicillin/streptomycin (100 μg/mL) for 3 h prior to stimulation with HKCA (6.25 × 10⁵cells/mL) or curdlan (100 μg/mL) at 37°C for 0–20 min. Cells were lysed (1% Triton, 120 mM NaCl, 50 mM Tris, 0.1% SDS, 1 mM EDTA, containing protease/phosphatase inhibitors) and resolved in SDS‐PAGE gels and transferred to PVDF membranes, blocked (Tris, 5% BSA, 0.05% Tween20) and probed with the indicated antibodies; pSyk (clone; C87C1), Syk (clone; D1I5Q), pErk and Erk (mAb Rabbit IgG), Pp38 (clone; D3F9), p38 (clone; D13E1), IκBα (clone; 44D4), pIκBα (clone; 14D4) (all immunoblotting antibodies from Cell Signaling Technologies) followed by anti‐rabbit‐HRP (Dako) secondary antibody. Proteins visualized by SuperSignal chemiluminescent reaction (Pierce) in a ChemiDoc station (BioRad). Densitometry measurements were performed with ImageJ software.

Total protein was isolated from cultured cells using RIPA buffer with added protease (Roche, 06538282001) and phosphatase inhibitors (Fisher Scientific, 502306746). Protein concentrations were quantified using a BCA assay (Pierce, PI23225) and equal amounts of protein were loaded for SDS-PAGE. GAPDH (Fitzgerald Industries International, 10R-G109a, 1:5000 dilution), p21 (Abcam, ab109199, and 1:500) and Erbb2 (Abcam, ab16901, 1:1000) primary antibodies and Anti-mouse IgG HRP-linked (Cell Signaling, 7076S) and Anti-rabbit IgG HRP linked (Cell Signaling, 7074S) secondary antibodies were used for blotting. Western blots were imaged after enhanced chemiluminescence using a Chemidoc station (Biorad).

Purified protein fractions were isolated using the SurePrep^™ RNA/Protein Purification kit (Fisher BioReagents, Fair Lawn, NJ, USA) and aliquots of protein (20 μg) were boiled for 5 min in Laemmli SDS loading buffer (10% glycerol, 5% SDS, 5% β-mercaptoethanol, 0.01% bromophenol blue and 125 mM TRIS-HCl [pH 6.8]) prior to loading onto a 12% acrylamide gel (Bio-Rad Laboratories, Hercules, CA, USA). The proteins were transferred to a PVDF membrane (Immobilon-P, Millipore, Bedford, MA, USA) using a mini Trans-Blot Electrophoretic transfer cell (Bio-Rad Laboratories, Hercules, CA, USA), which were then blocked with 5% non-fat milk. The membranes were probed overnight at 4°C with the anti-FAAH antibody (see Table 2) and then for 2 hours at room temperature with an ECL^™ Horseradish Peroxidase-linked whole secondary antibody (GE Healthcare UK Limited, Buckinghamshire, UK) diluted 1:5000. Reactive bands were detected by chemiluminescence with the Amersham^™ ECL^™ Prime Western Blotting Detection Reagent (GE Healthcare UK Limited, Buckinghamshire, UK) and the membranes were analyzed on a ChemiDoc station with Quantity one software (Bio-Rad Laboratories, Madrid, Spain). The optical density of the specific protein bands were assessed relative to the housekeeping protein GAPDH, and they were normalized to the controls.

293T-wt and 293T-gB cells were re-suspended in PBS and maintained on ice. Cell lysates were produced by sonication and the protein concentration was determined with Bicinchoninic acid assay (BCA). One microliter corresponding to 1 μg of cell lysate was spotted onto strips of nitrocellulose membranes (GE Healthcare, Little Chalfont, UK) and air-dried for 10 min. Five percent milk/PBS/0.05% Tween (Merck Millipore) was used to block the membrane for 30 min at room temperature. Plasma from mice was added to the membrane strips which were incubated overnight at 4°C and washed 3x with PBS/0.05% Tween for 10 min at RT. Anti-human IgG-HRP (Roth, Karlsruhe, Germany) (1:500) was added for 1 h at RT. Next, the membrane was washed 4x with PBS/Tween for 10 min at room temperature (RT). After incubation with HRP chemiluminescent substrate (ThermoFisher), signals were measured with a Chemidoc station (Bio-Rad, Hercules, CA). ImageJ software (NIH, freeware) was used for signal quantifications of dot-blots.

Cultured cells were treated with PB or Lunasin (100 μM) for 24 hours. Acid extraction of histones was performed as described [79 (link)]. 10 μg of total purified histones were loaded per lane and run on 15% gels (Lonza). Cells were harvested and re-suspended in RIPA buffer (Sigma). Protein concentrations of cell lysates were determined by a bicinchoninic acid assay (Thermo Fisher Scientific) and 20–60 μg of total protein was loaded per lane on 10% gels (BioRad), subjected to SDS-PAGE, and transferred to a PVDF membrane (EMD Millipore). Lysates were probed with antibodies that recognize phosphorylated AKT (Cell Signaling #9916S), phosphorylated FAK (Cell Signaling #9330S), phosphorylated ERK 1/2 (Cell Signaling #4094), β-Actin (Santa Cruz #sc-47778), H3K9 (EMD Millipore #07-352), H4K12 (EMD Millipore #04-119), H4K8 (EMD Millipore #07-328), H3K14 (EMD Millipore #07-353). Densitometry and image analysis were performed using a ChemiDoc station equipped with ImageLab software (BioRad).