Complete protease inhibitor and phosphatase inhibitor

Cells were homogenized in RIPA buffer (ThermoFisher, United States) containing complete protease inhibitor and phosphatase inhibitors (Roche), and a BCA assay kit (Beyotime, China) was used to determine total protein concentration. Then, samples were further diluted in 5× Loading buffer (Beyotime, China) and boiled for 30 min. Equal amounts of protein (20 μg of total protein) from each sample were loaded and ran on 8–16% Expressplus PAGE gels (Genscript) and transferred to PDVF membranes (Merck, United States). After blocking in QuickBlock Blocking Buffer (Beyotime, China) at room temperature, the membranes were probed with primary antibodies overnight at 4°C respectively. A control blot incubated with β-actin antibody was used to normalize the amount of protein. The membrane was washed and then probed with HRP-linked anti-rabbit secondary antibody for 1 h at RT, followed by ECL detection (Merck, United States). The intensities of the bands were scanned and measured with Fiji (Schindelin et al., 2012 (link)). Specifically, phos binding reagent acrylamide was added to PAGE gels in concentration of 50 μM.

Proteins were extracted from primary epithelial cell cultures and cell lines in RIPA buffer as previously described [60 (link), 61 (link)]. For immunoprecipitation, cells were suspended in 1% triton-X100 containing Phosphate-Buffered Saline (PBS) and Sodium dodecyl Sulfate (SDS). Cell lysate (4 mg total protein) was made by boiling samples at 100 °C followed by sonication. Lysate was precleared by incubating with 50 µl of protein A+G agarose beads (Fisher Scientific) for 1 h at 4 °C. 2 µg of Anti-Ubiquitin antibody (Santa Cruz, sc#8017) or anti-DLL1 antibody (Abcam, ab84620) or respective species specific control antibodies were incubated overnight with 4 mg of proteins. At the time of antibody addition, complete protease inhibitor and phosphatase inhibitors (Roche) were added besides 1 mM Dithiothreitol (DTT). Lysates were incubated overnight with the antibodies followed by three washes with triton-X100 containing PBS and one wash with 20% sucrose in PBS. Western blot analysis was performed using the standard protocol.

For purifying Flag-tagged CCN2 fusion proteins, HEK-293 T cells with Flag-tagged CCN2 stable overexpression were harvested in RIPA lysis buffer supplemented with complete protease inhibitor and phosphatase inhibitor (Roche Applied Science). The lysate was cleared by centrifugation at 12000 g before being loaded to M2 anti-Flag mAb agarose beads (Sigma, St. Louis, MO, USA) pre-equilibrated in RIPA buffer overnight at 4 °C. The beads were washed with RIPA buffer five times and 30ul RIPA buffer was added to cover the beads. The RIPA buffer containing beads and Flag-tagged CCN2 fusion proteins were stored at 4 °C for the next experiment. For the extraction of membrane proteins, MHCC97H cells were grown to 75% confluence. The membrane proteins were extracted using a ProteoExtract Native Membrane Protein Extraction Kit (M-PEK Kit; Calbiochem, La Jolla, CA, USA) according to manufacturer’s instruction. For the binding assay, Flag-tagged CCN2 bind to the beads was incubated with membrane proteins with or without LMWH (2 μg/ml, Santa Cruz Biotechnology) overnight at 4 °C in RIPA buffer. Then, the beads were washed with RIPA buffer five times and bound proteins eluted using Flag peptide (Sigma). The washed protein was boiled in loading buffer, resolved on SDS-PAGE. Subsequent immunoblots were probed with the appropriate antibody and detected by ECL.