Enhanced chemiluminescence substrate kit

Cells were washed with ice‐cold PBS and lysed on ice in Radio‐Immunoprecipitation Assay (RIPA) buffer (Sigma‐Aldrich, St. Louis, MO, USA) with Protease Inhibitor Cocktail (Sigma‐Aldrich). For preparation of lysate from tissues, frozen tissues were sliced using a clean razor blade and thawed in RIPA buffer containing protease Inhibitors. Equal amounts of proteins (30 μg) were loaded into the wells of the SDS‐PAGE gel and then transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% non‐fat milk in TBST for 1 hr. After blocking, the membranes were incubated with the respective primary antibodies overnight at 4°C. The primary antibodies used were as follows: rabbit anti‐GAPDH antibody (Ab; Santa Cruz Biotechnology, Santa Cruz, CA, USA), or rabbit anti‐JAG1 Ab (Santa Cruz Biotechnology), mouse anti‐HES1 Ab (Santa Cruz Biotechnology), rabbit anti‐HES5 Ab (Abcam, Cambridge, UK) and rabbit anti‐NICD Ab (Millipore). After washing, membranes were incubated in PBS with 0.1% Tween with antimouse or anti‐rabbit IgG secondary antibody conjugated with horseradish peroxidase (Santa Cruz Biotechnology) for 1 hr. Blots were detected using enhanced chemiluminescence substrate kit (GE Healthcare, Piscataway, NJ, USA).

Western blots were performed as previously described [54 (link)]. The following antibodies were used: IGFBP5 (ab4255, Abcam, Cambridge, UK), vimentin (ab8069, Abcam), GAPDH (ab75834, Abcam), HIF1α (NB100-134, Novus, St. Louis, MO, USA), IGF1R-P(Try1135/1136)(3024, Cell Signaling Technology (CST), Danvers, MA, USA), ERK1/2-P (Thr202/Tyr204) (4376, CST), ERK1/2 (4695, CST), E-cadherin (3195, CST), p38-MAPK-P (Thr180/Tyr182) (9215, CST), p38-MAPK (9212, CST), sheep anti-mouse IgG (ZB-2305, ZSGB-Bio, Beijing, China), and sheep anti-rabbit IgG (ZB-2301, ZSGB-Bio). Enhanced chemiluminescence substrate kit (RPN2232, GE Healthcare, Piscataway, NJ, USA) was used for the chemiluminescent detection of signals with BioMax film (Kodak, Rochester, NY, USA).

Cells were lysed on ice with RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA) containing protease inhibitor cocktail (Sigma-Aldrich). Protein content of the lysates was determined using the Bradford Protein Assay kit (Bio-Rad, Hercules, CA, USA). Equal amounts (40 μg/lane) of protein were separated by 12 % SDS PolyAcrylamide Gel Electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The blots were incubated with rabbit anti-PCNA antibody (Ab), rabbit anti-phospho-STAT1 (Tyr701) Ab (Cell Signaling Technology, Beverly, MA, USA), rabbit anti-S100A4 Ab (Abcam, Cambridge, UK), mouse anti-β-actin Ab (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-fibronectin Ab mouse anti-β-catenin Ab (Santa Cruz Biotechnology), mouse anti-STAT1 Ab, and mouse anti-c-fos Ab (Abcam). Primary Ab binding was detected with secondary antibody (anti-mouse or anti-rabbit HRP-conjugated IgG secondary antibody) that was detected using enhanced chemiluminescence substrate kit (GE Healthcare, Piscataway, NJ, USA). Quantification was performed with a ChemiDoc TM XRS + scanner and Image Lab Software (Bio Rad, CA, USA). The densities of each sample were normalized to the β-actin.