Coomassie blue

MBP and GST fusion proteins were affinity purified in column buffer (20 mM Tris (pH 7.0), 150 mM NaCl, 2 mM EDTA, and 0.1% NP40) with Amylose Resin (NEB) or GST-bind resin (Millipore), respectively. MBP fusion proteins were eluted with maltose and GST fusion proteins were eluted with Glutathione per manufacturers recommendations. For binding reactions, 1 μg of each protein was incubated with 50 μl GST-bind resins in NP40 buffer for 1 hr. Beads were washed 3× with NP40 buffer before 50 μl of 5× Sample Buffer was added. Proteins were boiled for 10 min and resolved by SDS-PAGE on a 4–12% Bis-Tris gel (ThermoFisher). Protein gels were stained with Coomassie Blue (Biorad) for 30 min and destained for 2–3 h in destain buffer (40% MeOH and 10% Acetic Acid) before imaging. For in vivo pull-down assays, purified recombinant proteins were left on beads and incubated with protein lysates in NP40 buffer for 2 h. Beads were washed 3× with NP40 buffer before adding 50 μl of 5× sample buffer. Proteins were separated by SDS-PAGE and immunoblotted with anti-VCP (Cell Signaling, 7F3, Rabbit mAb #2649) and anti-GFP (mouse monoclonal, Invitrogen clone 3E6; A-11120). Total lysates were immunoblotted with anti-tubulin (E7-c, DSHB). HRP conjugated goat anti-mouse and goat anti-rabbit secondary antibodies (ThermoFisher) were used at 1:2000.

The presence of MMP-2 and MMP-9 media was determined by gelatin zymography as described previously^{23 (link)}. In brief, culture supernatants were obtained from liver cells cultured under basal conditions or with media derived from C26 cell cultures, pretreated or not with sICAM-1. Cell culture supernatants were collected and run in 1% gelatin containing 10% bis-acrylamide gel. Gels were incubated overnight in developing buffer and stained with Coomassie Blue (BioRad, CA, USA). For uPA quantification, zymographies were carried out in gels containing gelatin and plasminogen. Images were taken through Quantity One program (BioRad, CA, USA).

The bladder cancer EJ cells cultured in 6-well plates were treated with various concentrations of SPHF for 24 h. The conditioned medium was electrophoresed on a polyacrylamide gel containing 1 mg/mL gelatin (Sigma-Aldrich, St. Louis, MO, USA). The gel was then washed at room temperature for 2 h with 2.5% Triton X-100 and maintained at 37 °C overnight in a pH 7.5 buffer containing 10 mM CaCl₂, 150 mM NaCl, and 50 mM Tris-HCl (Sigma-Aldrich, St. Louis, MO, USA). The gel was stained with 0.2% Coomassie blue (Bio–Rad Laboratories, Hercules, CA, USA) and images were captured using a lightbox (Matin International, Seoul, Korea). Proteolysis was detected as a white area in a dark blue field with the ImagePro Plus 6.0 software (Media Cybernetics, Bethesda, MD, USA).

All female samples were run on one gel under the same conditions; all male samples were run on another gel under the same conditions. Western immunoblotting was conducted as described previously [6 (link)].
Membranes were stained overnight using primary antibodies against γH2AX (1:500, Cell Signaling, Danvers, MA), TBX18 and EAAT2 (1:1000, Abcam, Toronto, ON), and actin (1:2000, Abcam, Toronto, ON). Primary antibody binding was detected using horseradish peroxidase-conjugated secondary antibodies and the Enhanced Chemiluminescence Plus System (Amersham Biosciences, Baie d'Urfe, Quebec). Chemiluminescence was detected using a FluorChem HD2 camera with FluorChem software (Cell Biosciences); gel images were saved and processed using Adobe Professional under the same conditions. Bands corresponding to antibody binding in all samples were carefully cropped; no images were spliced. The membranes were stained with Coomassie blue (BioRad, Hercules, CA) to confirm equal protein loading. Signals were quantified using the NIH Image J64 software and normalized relative to actin or Coomassie staining.

The protein profile of PPE, Ara h 1, Ara h 2, Ara h 3 and Ara h 6 was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing and reducing conditions. Each protein sample was dissolved in PBS (137 mM NaCl, 3 mM KCl, 8 mM Na₂HPO₄, 1 mM KH₂PO₄, pH 7.2) and mixed 1:1 (v/v) with 2x Laemmli sample buffer (161-0737, Bio-Rad, Hercules, CA, US) with the addition of 2 M dithiothreitol (348-12-3, Sigma-Aldrich, Darmstadt, Germany) for reducing conditions, and subsequently heated for 5 min at 95°C. Five micrograms of protein/well as well as 3 µL of molecular marker (161-0363, Bio-Rad) were loaded onto Mini Protean TGX Precast Protein 4-20% Gels (4568094, Bio-Rad) and electrophoresed on a Mini-PROTEAN Tetra Cell (Bio-Rad) filled with 10X Tris/Glycine/SDS electrophoresis buffer (161-0732, Bio-Rad) (1:10 v/v) prepared according to manufacturer’s protocol. The gels were stained with Coomassie Blue (161-0786, Bio-Rad) at room temperature (RT) for 3 h, and subsequently destained with MQ water at RT overnight. Destained gels were photographed using Imager ChemiDoc XRS+ (Bio-Rad).

Samples were run in 6 % polyacrylamide (Biorad) gels under reducing, denaturing conditions. Staining was performed with Coomassie blue (1 g Coomassie R250 in 40 % methanol) or silver (Biorad; volume used was half of manufacturer’s recommendation). Gels containing collagen fluorescently labelled with Alexa 647 (see below) were imaged with a gel scanner (Typhoon 9410 Gel and Blot Imager). For Western blotting, samples were transferred to 0.22 μm PVDF membranes (Biorad) and probed for the presence of procollagen with a collagen II specific monoclonal antibody (5B2.5, Abcam), which recognizes the sequence GGFDEK in the N-terminal telopeptide.