Imagequant las 4000 analyzer

To determine soluble CD147 in CCA-conditioned media, three CCA cell lines, namely KKU-055, KKU-100, and KKU-213A, were seeded and cultured for 24 h. After media were replaced, cells were cultured for another 48 h until they reached cell confluence. CM was centrifuged at 2000× g for 5 min at 4 °C to remove cell debris. CM was concentrated to reduce the volume to 1/50 time by Vivaspin^®20 (GE Healthcare, Buckinghamshire, UK). The concentrated CM was immediately used for Western blotting.
Total protein preparation and Western blotting were performed as previously described [17 (link)]. Protein concentrations were determined using Bradford reagent (Bio-Rad Laboratories, CA, USA). The immunoreactivity was detected using the ECL^TM Prime Western Blotting Detection System (GE Healthcare, Buckinghamshire, UK). The signals were visualized by ImageQuant™ LAS4000 analyzer and ImageQuant TL software ((version 8.0), GE Healthcare, Uppsala, Sweden).

Proteins were separated in 10–12% polyacrylamide gel and then transferred onto polyvinylidene difluoride membranes (Millipore). The membranes were blocked for 30 min using 10 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.05% Tween-20, and 5% low-fat milk powder. The membranes were incubated with primary antibodies, followed by secondary antibodies conjugated to HRP. The proteins were visualized by chemiluminescence using an ImageQuant LAS4000 analyzer (GE Healthcare). Uncropped images are shown in Supplementary Fig. 9.

For the Western blot analysis, cells were harvested and lysed in protein lysis buffer (Merck Millipore, St. Louis, MO, USA) supplemented with a complete protease inhibitor cocktail. Cell lysates were prepared in a previously described manner [40 (link)]. Primary antibodies against PAR1 (ATAP2:sc-13503; Santa Cruz Biotechnology, Dallas, TX, USA), RhoA, ROCK1, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signaling, Danvers, MA, USA), and β-actin were purchased from Novus Biologicals (Littleton, CO, USA), both GAPDH and β-actin were used as loading controls. Appropriate HRP-conjugated secondary antibodies were used to detect proteins using a luminol-based enhanced chemiluminescent (ECL) substrate (Thermo Scientific). An ImageQuant LAS 4000 analyzer (GE Healthcare Life Science, Pittsburgh, PA, USA) was used to detect protein expression levels.

Cell lysates from WRO cells stably transfected with either ABI3 or control (empty vector) were incubated with Human Phospho-Kinase Array Kit (cat. # RY003), which detects relative phosphorylation levels of 46 intracellular serine/threonine/tyrosine kinases, and Human Apoptosis Array Kit (cat# ARY009), which detects levels of 35 apoptosis-related proteins (R&D Systems, Minneapolis, MN). The intensity score of each duplicated spot was measured using ImageQuant LAS4000 Analyzer (GE Healthcare, Chicago, IL) and quantified using ImageQuant TL software (GE Healthcare). The averaged intensity was calculated by subtracting the averaged background signal, according to the manufacturer's instructions. Fold changes were calculated based on the average density values of each protein expressed from WRO ABI3 expressing cells divided by the average density values of control cells.

After treating LFs with 25 µg mL^-1 GNP-HCIm and 10 µM Imatinib for 24 h, cells were lysed. Proteins (10 µg) were loaded onto SDS-PAGE and transferred onto a PVDF membrane using a Trans Blot turbo system (Bio-Rad). After 1 h incubation in 5% Bovine Serum Albumin (BSA) diluted in PBS and three washes with PBS containing 0.1% Tween 20 (TBST), the membrane was incubated overnight with anti-c-Abl (phospho Y412) antibody (ab4717, Abcam) at a 1:1000 dilution in 1% BSA. After three washes with TBST (10 mL), membranes were incubated with Rabbit anti-Mouse IgG H&L (HRP) (ab6728, Abcam) secondary antibody at a 1:2000 dilution in 1% BSA in TBST, for 1 h at room temperature. Clarity Western ECL (Bio-Rad) solution was used according to the provided protocol. The same procedure was applied to identify c-Abl by anti-c-Abl antibody (ab15130, Abcam) at a 1:100 dilution and β-actin (15G5A11/E2, Cat #MA1-140, Thermo Fisher Scientific) at a 1:5000 dilution. Also in this case we used as secondary antibody Rabbit anti-Mouse IgG H&L (HRP) (ab6728, Abcam). All immunoblots were acquired with the ImageQuant LAS 4000 analyzer (GE Healthcare).

Ten micrograms of proteins were precipitated by addition of 1.22 M trichloroacetic acid (TCA) and the pellet recovered after centrifugation was submitted to sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE). Protein bands were transferred onto a Millipore polyvinyldivinyl fluoride (PVDF) membrane (Billerica, MA, USA) by using a trans blot turbo system (BioRad). After 1 h incubation in 5% milk diluted in phosphate buffer saline (PBS) and three washes with phosphate buffer saline containing 0.1% Tween 20 (PBST), the membrane was incubated overnight with AAT antibody (ab9400; Abcam, Cambridge, UK) at a 1:2500 dilution in 1% milk. The membrane was washed three times with PBST (10 mL), incubated with the secondary antibody, rabbit anti-mouse IgG H&L (HRP) (ab6728, Abcam), at a 1:2000 dilution in 1% milk in PBST, for 1 h at room temperature. The membrane was washed again (three times) with PBS and incubated in ECL Westar ηC Ultra (Cyanagen, Bologna, Italy) solution according to the provided protocol. The same procedure was applied for the identification of free and complexed HNE by using the anti HNE antibody (PA5-29659, Thermo Scientific) at a 1:1000 dilution.
All immunoblots were acquired with the ImageQuant LAS 4000 analyzer (GE Healthcare Chicago, IL, USA).

Ten micrograms of proteins were precipitated by the addition of 1.22 M TCA and the pellet recovered after centrifugation was submitted to SDS-PAGE. Protein bands were transferred into a Millipore polyvinyl divinyl fluoride (PVDF) membrane (Billerica, MA, USA) by using a trans blot turbo system (BioRad Laboratories, Segrate (MI), Italy). After 1 h incubation in 5% milk diluted in phosphate buffer saline (PBS) and three washes with phosphate buffer saline containing 0.1% Tween 20 (PBST), the membrane was incubated overnight with AAT antibody (ab9400; Abcam, Cambridge, UK) at a 1:2500 dilution in 1% milk. The membrane was washed three times with PBST (10 mL), incubated with the secondary antibody, rabbit anti-mouse IgG H&L (HRP) (ab6728, Abcam), at a 1:5000 dilution in 1% milk in PBST, for 1 h at room temperature. The membrane was washed again (three times) with PBS and incubated in ECL Westar ηC Ultra (Cyanagen, Bologna, Italy) solution according to the provided protocol. The same procedure was applied for the identification of free and complexed HNE by using the anti HNE antibody (MA5-2548, Invitrogen, Waltham, MA, USA) at a 1:1000 dilution and secondary antibody anti-rabbit (ab6721, Abcam) at a 1:3000 dilution. All immunoblots were acquired with the Image Quant LAS 4000 analyzer (GE Healthcare, Chicago, IL, USA).