Biomax light film

The cellular and EV proteins were extracted with RIPA buffer [36 (link)], and equal amounts of protein extracts (protein concentration measured by BCA assay) were subjected to SDS-PAGE analysis, transferred onto PVDF membranes, and incubated at 4 °C with specific antibodies overnight (Table S1). Specific HRP-conjugated secondary antibodies were used, and protein bands were detected using an enhanced chemiluminescence kit and Kodak BioMax Light Film (Eastman Kodak, Rochester, NY, USA).

Cell monolayers were washed, scrapped and lysed in buffer [50 mM Tris-HCl (pH 7.6), 0.5 M NaCl, 0.02% NaN₃, 0.6% NP40, 5 mM EDTA, 1 mM iodoacetamide, 1 mM PMSF]. The protein concentration was determined by Bradford method [43 (link)]. When needed, the protein extracts were incubated for 24 h at 37°C with 100 nM lumican. Total cell proteins (10 μg) were subjected to electrophoresis in a 0.1% SDS, polyacrylamide gel and proteins were transferred onto PVDF membranes (Millipore). The membranes were blocked in 5% non fat milk (BioRad) for 2h at RT, washed with TBS/0.1% Tween 20 and the membranes were incubated overnight at 4°C with the following primary antibodies: anti-human Snail (#3879, Cell Signaling Technology, 1:500), anti-actin (#sc-1616, Abcam, 1:500) and anti-MMP-14 (#ab38971, Abcam, 1:5000). After washing and incubation with corresponding HRP-secondary antibody, bands were visualized using the ECL Plus Chemoluminescence Detection kit (GE Healthcare, Orsay, France or Thermo Scientific, Waltham, MA, USA). Membranes were either developed with Kodak BioMax Light Film (Eastman Kodak, Rochester, NY, USA) or scanned on a Chemidoc (Biorad, Marne la coquette, France) imaging and gel documentation system. The protein extracts and the control lysate from human SNAIL transfected 293T cells (Santa Cruz Biotechnology) were used as a control.

Cell extracts were prepared as described previously [28 (link)]. Briefly, exponentially growing cells were lysed with M-PER Mammalian Protein Extraction Reagent supplemented with Halt Protease Inhibitor Cocktail according to the manufacturer’s instructions. The concentration of proteins in each sample was quantified with the BCA Protein Assay Kit according to the manufacturer’s protocol. Protein lysates (30 µg), immunoprecipitate, or tubulin fraction extracts were separated on 10% SDS-PAGE gels (Bio-Rad, CA, USA) and electroblotted (120 min; 200 mA; 4 °C) onto nitrocellulose membranes (Bio-Rad). The membranes were blocked with 5% BSA in PBS (2 h; RT), incubated with primary antibodies (overnight; 4 °C), and after washing incubated with horseradish peroxidase-conjugated secondary antibodies (1 h; RT). Chemiluminescence detection proceeded with Pierce ECL Western Blotting Substrate and Kodak BioMax Light Film (Eastman Kodak, Rochester, NY, USA). Protein bands were scanned (HP Scanjet G4050) and quantified using Gel Doc 2000 gel documentation system (Bio-Rad).

Cells were lysed in RIPA buffer (100 mM Tris–HCl, pH 7.5, 300 mM NaCl, and 0.2% SDS) containing 1% IGEPAL CA-630 and the Halt™ Protease Inhibitor Cocktail (Thermo Fisher Scientific) with subsequent centrifugation (18,000 × g, 4 °C, 20 min). The total protein concentration in the supernatants was quantified using the BCA method (Pierce BCA Protein Assay; Thermo Fisher Scientific). Samples with equal total protein content were loaded and separated on SDS–PAGE gels (Mini-PROTEAN TGX Stain-Free Gels; Bio-Rad Laboratories, Hercules, CA, USA) and then transferred onto nitrocellulose membranes (Bio-Rad Laboratories). We used the following primary antibodies: rabbit anti-NMU, rabbit anti-NMUR1 (Genetex, Irvine, CA, USA), mouse anti-IGFBP-7 and mouse anti-vimentin (Santa Cruz Biotechnology, Dallas, TX, USA). For the analysis of ERK1/2 levels, mouse anti-ERK1/2 (Santa Cruz Biotechnology) and rabbit anti-pERK1/2 antibodies (Thermo Fisher Scientific) were used. GAPDH or α-tubulin was used as a loading control and detected using rabbit anti-GAPDH or mouse anti-α-tubulin antibodies, respectively (Abcam, Cambridge, GB). HRP-conjugated goat, anti-mouse (Santa Cruz Biotechnology) or anti-rabbit secondary antibodies (Thermo Fisher Scientific) were used. The signal was detected by measuring the chemiluminescence (Thermo Fisher Scientific) with Kodak BioMax Light Film from Eastman Kodak (NY, USA).

Proteins isolated from HT-29 cells were extracted with NP-40 lysis buffer (50 mM Tris, pH 8.0, containing 1% Nonidet-Igepal, 150 mM NaCl, 5 mM EDTA) with the Halt protease inhibitor cocktail (Thermo Scientific, Waltham, MA, USA), and the soluble protein fraction was collected through centrifugation. The protein concentrations in the cell lysates were measured with the BCA method (Pierce/Thermo Scientific, Waltham, MA, USA) and were equalized between samples. The protein extracts were subjected to SDS-PAGE analysis and were electro transferred onto PVDF or nitrocellulose membranes (BioRad, Hercules, CA, USA) followed by immunodetection goat anti human ABCC4 #PA5-18315 (Thermo Fisher Scientific), rabbit anty human ABCG2 #ORB155559 (Biorbyt). The control-mouse rabbit anti-α-tubulin antibody conjugated with HRP (NB100-690H) was obtained from Novus Biologicals (Centennial, CO, USA) and used as a loading control. Detection was performed using secondary HRP-conjugated antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) followed by incubation with an enhanced chemiluminescence kit (Thermo Scientific, Waltham, MA, USA) and development with Kodak BioMax Light Film (Eastman Kodak, Rochester, NY, USA).

Stimulated HBSMCs were harvested with a cell scraper and were solubilized in a lysis buffer consisting of 20 mM Tris–HCl (pH 7.5), 1 % Nonidet P-40, 1 mM EDTA, 50 mM NaF, 50 mM sodium β-glycerophosphate, 0.05 mM Na₃VO₄, 10 μg/ml leupeptin, and 100 μM phenylmethylsulfonyl fluoride. Following centrifugation at 5000g for 5 min, the resultant supernatant was used as the lysate after protein concentration determination using the Bradford assay (Bio-Rad, Hercules, USA). The lysates were resolved in a 10 % SDS polyacrylamide gel and were electrotransferred to Hybond-P Polyvinylidene difluoride membrane (GE healthcare, Amersham Place, UK). Immunodetection of JNK1 and phosphorylated JNK1 was performed using anti-JNK1 (1:1000 dilution; Cell Signaling, Massachusetts, USA), and anti-phosphorylated JNK1 (Thr183/Tyr185) antibodies (1:1000; Cell Signaling), respectively. The signal was detected by incubation with an anti-rabbit secondary antibody conjugated to horseradish peroxidase (1:10,000; Promega, Tokyo, Japan), followed by chemiluminescence detection using the SuperSignal kit (Pierce Chemical Company, Rockford, USA) and Kodak BioMax light film (Kodak, Tokyo, Japan). The densities of the bands were quantified using the computer software, ImageJ (developed at the U.S. National Institutes of Health).