Myecl imager

Preparation of cell lysates from cells was carried out as previously described [75 (link)]. Briefly, cells were washed twice with cool PBS and lysed at 4 °C with lysis buffer (10 mM Tris–HCl (pH 7.6), 50 mM NaCl, 30 mM sodium pyrophosphate, 5 mM EDTA, 5 mM EGTA, 0.1% SDS, 1% Triton X-100, 50 mM NaF, 0.1 mM Na3VO4, 1 mM PMSF, 1 mM benzamidine, 1 mM iodoacetamide, and 1 mM phenantroline). Cell lysates were obtained by centrifugation at 21,380× g for 30 min at 4 °C; protein concentration in the supernatant was determined by BCA protein assay (Cat no. 23227, Pierce, Rockford, IL, USA), and lysates were adjusted to equivalent concentrations with lysis buffer. Aliquots of 10–40 µg of total cell lysate were then separated on SDS–PAGE. Proteins were transferred to PVDF membranes (Amersham™ Hybond^® P Western blotting membranes, PVDF, Cta no. GE10600021, Sigma, St. Louis, MO, USA) that were blocked overnight at 4 °C with 5% nonfat milk in TTBS (TBS with 0.05% Tween-20). Incubation was first was carried out overnight with primary specific antibodies (Table S1) at 4 °C and then with horseradish peroxidase-conjugated secondary antibodies in blocking solution for 1 h at room temperature. Immunoreactive bands were visualized by ECL using a MyECL^TM Imager (ThermoFisher Scientific, Waltham, MA, USA). Uncropped and unmodified images of immunoblots are provided in Figure S4.

CRC cells were harvested after 48 hours of culture in the presence of drugs or control vehicle, lysed by using 1X cell lysis buffer containing 1mM PMSF. Cell lysates were subjected to PathScan RTK Signaling Antibody Array Kit (Cell Signaling Technology, Danvers, MA) (Chemiluminescent Readout) as per manufacturer's protocol. Briefly, the PathScan RTK Signaling Antibody Array Kit is a slide-based antibody array founded upon the sandwich immunoassay principle. The array kit allows for the simultaneous detection of 28 receptor tyrosine kinases and 11 important signaling nodes when phosphorylated at tyrosine or other residues. Target-specific capture antibodies using biotinylated protein as positive control, and nonspecific IgG as negative control, were spotted in duplicate onto nitrocellulose-coated glass slides. Cell lysates were then incubated on the slide followed by a biotinylated detection antibody cocktail. Streptavidin-conjugated HRP and LumiGLO reagent were then used to visualize the bound detection antibody by chemiluminescence. An image of the slide was captured with myECL^TM Imager (ThermoFisher Scientific, Frederick, MD). Intensities of spots were quantified using myImage analysis software (ThermoFisher Scientific, Frederick, MD).

For Western blot analysis, mouse livers and HSC were harvested in RIPA-lysis buffer containing protease and phosphatase inhibitors. Protein extraction was performed via sonication followed by centrifugation. After colorimetric protein quantification with DC protein assay (Bio-Rad Laboratories, Düsseldorf, Germany) according to the manufacturers’ instructions, samples of equal protein amount (Col-GFP 40 µg/well, LX-2 20 µg/well, primary HSC 3 µg/well, tissue 100 µg/well) were prepared by adding 1 M dithiothreitol (DTT-1M) and NuPAGE-LDS electrophoresis sample buffer (Thermo Fischer). For cell lysate supernatant analyses, 45 µL of each supernatant was mixed with LDS sample buffer (4×) and DTT-1M. Preparation of samples for Western blot analysis was performed as described previously [20 (link)]. Primary and secondary antibodies used are listed in Supplementary Table S1. Visualization was performed using the SuperSignal West Dura Extended Duration Substrate and the myECL TM imager (both from ThermoFisher).