Protease inhibitor and phosphatase inhibitor

Total proteins were extracted using RIPA lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5% sodium-deoxycholic acid, 0.1% sodium dodecyl sulfate, 1% Triton X-100 and 2 mM EDTA) containing 1% protease inhibitor and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Protein concentrations were calculated using a DC protein assay kit (Bio-Rad, Hercules, CA, USA). Equivalent amounts of protein were subjected to electrophoresis on a 10% sodium dodecyl sulfate-polyacrylamide gel, and the separated proteins were electrotransferred onto polyvinylidene fluoride membranes (Bio-Rad). The membranes were blocked with blocking buffer containing 5% bovine serum albumin (BSA; Bioshop, South Korea) for 1 h and then incubated at 4°C overnight with primary antibodies (COX-2, iNOS, and β-actin; Cell Signaling Technology, Danvers, MA, USA). Membranes were washed with Tris-buffered saline Tween-20 (TBST; 20 mM Tris, pH 7.4, 150 mM NaCl and 0.1% Tween-20) and then incubated for 1 h at room temperature with secondary antibodies diluted in TBST (horseradish peroxidase [HRP]-anti-rabbit and HRP-anti-mouse; AB FRONTIER, Seoul, South Korea). The membranes were washed and then developed with a Clarity Western ECL substrate kit (Bio-Rad), and images were captured using a ChemiDoc XRS+ Imager (Bio-Rad).

Cells were lysed with RIPA buffer supplemented with protease inhibitor and phosphatase inhibitors (all Sigma Aldrich). After 30 minutes incubation on ice with periodic pulse vortexing, cell‐lysates were centrifuged at 16 000 g for 15 minutes at +4°C. For an equal loading, protein concentration was measured using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to manufacturing instructions. A 4%‐12% SDS‐PAGE (Bio‐Rad, Hercules, CA, USA) was used to separate proteins following a transfer onto PVDF membranes (GE Healthcare). The membrane was blocked with bovine serum albumin; BSA (Sigma Aldrich) and the following antibodies were used for incubation over night: phospho‐Smad3 (1:2000, Anti‐Smad3 PhosphoS423+S425, Abcam, Cambridge, UK), total Smad2/3 (1:1000 R&D Systems), LC3 A/B (1:1000 D3U4C, Cell Signaling Technology, Massachusetts, USA), and Pan‐Actin (1:2000, D18C11, Cell Signaling Technology). Protein bands were visualized using HRP‐linked anti‐rabbit (Cell Signaling Technology) or anti‐goat (Abcam) and SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific).

Tissues and harvested cells were lysed and ultrasonicated in RIPA reagent containing protease inhibitor and phosphatase inhibitor (Sigma-Aldrich) on ice for 30 min. The supernatant was collected after centrifugation at 14,000 × g for 10 min at 4 °C. The protein concentration was measured by bicinchoninic acid (BCA) assay. Protein extracts were resolved on 10% sodium dodecylsulfate-polyacrylamide (SDS-PAGE) gels (Wuhan Boster Biological Technology Ltd., Wuhan, China) prior to being transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). After blocking in 5% skim milk for 2 h, the membrane was incubated in primary antibody (Table S7) overnight at 4 °C and then incubated with secondary antibody: goat anti-rabbit IgG or goat anti-mouse IgG (Table S8) for 2 h at room temperature. Our bands were visualized with enhanced chemiluminescence kit (Thermo Scientific Fisher, Waltham, MA, USA) on a Tanon-5200 ECL imager (Tanon, Shanghai, China). The protein levels were quantified using Image J software. Target protein bands were normalized to the loading control GAPDH.

Using PBS, BCa cells were washed 3 times and then were lysed for 30 minutes on ice with a solution that contained RIPA buffer, protease inhibitor and phosphatase inhibitor (Sigma‐Aldrich). The cell lysates were centrifuged at 13 000 × g for 15 minutes, and a Bradford protein assay (Bio‐Rad) was used to determine the protein concentration of the collected supernatants. Western blot analysis was performed after the total protein samples were separated by 7.5%‐15% SDS‐PAGE. Immunoreactive bands were visualized with an enhanced chemiluminescence kit (Bio‐Rad) and were then detected with a Molecular Imager ChemiDoc XRS + Imaging system (Bio‐Rad). Table S2 and Table S3 list the primary antibodies and secondary antibodies used, respectively.