Protease and phosphatase inhibitor cocktail

Western blot analysis was undertaken as previously described (Lee et al., 2006). Whole-cell lysates were prepared in RIPA lysis buffer (Thermos Scientific, Waltham, MA, USA) supplemented with protease and phosphatase inhibitor cocktails (Gendepot, Katy, TX, USA). Total cell proteins were size-fractionated using SDS-PAGE and electro-transferred to Immobilon-P membranes (Millipore Corp., Bredford, MA, USA). Detection of specific proteins was carried out with enhanced chemiluminescence, following the manufacturer’s instructions (Amersham Biosciences, Piscataway, NJ, USA).

Western blot analysis was performed as previously described [34 (link)]. MH7A cells were treated with IP lysis buffer (Thermo Fisher Scientific, Rockford, IL, USA) containing protease and phosphatase inhibitor cocktails (GenDEPOT) following manufacturer protocol. Lysates were centrifuged at 16,609× g (Hanil, Incheon, Republic of Korea) and 4 °C for 15 min. Protein concentrations were measured using at Bicinchoninic Acid Protein Assay Kit (Thermo Scientific). Samples were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Darmstadt, Germany). Membranes were incubated overnight at 4 °C with primary antibodies phospho-Smad2/3 (1:1000, Cell Signaling, Danvers, MA, USA), phospho-Smad1/5/9 (1:1000, Cell Signaling), LC3B (1:1000; Cell Signaling), p62 (1:1000; Cell Signaling) and Beclin-1 (1:1000; Cell Signaling). Next, they were incubated with HRP-conjugated anti-rabbit (1:50,000; ENZO Life Science, Farmingdale, NY, USA) secondary antibodies for 1 h. An enhanced chemiluminescence kit (Amersham Pharmacia, Piscataway, NJ, USA) and ABI680 Analyzer (Amersham) were used to detect protein bands; signals were quantified in Image J. The loading control was β-actin antibody (1:5000; Sigma-Aldrich).

The cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (P3200-010; Biosesang, Gyeonggi-do, Korea) containing protease and phosphatase inhibitor cocktails (GenDepot, Barker, TX, USA). The proteins were resolved by sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) and transferred onto nitrocellulose membranes. The blots were immunostained with appropriate primary antibodies, followed by incubation with secondary antibodies conjugated to horseradish peroxidase. Antibody binding was detected with an enhanced chemiluminescence reagent (Bio-Rad. Hercules, CA, USA). Primary antibodies against the following molecules were used: ACSS2 (Thermo Fisher Scientific) and β-actin (Santa Cruz Biotechnology). The secondary antibodies were purchased from Santa Cruz Biotechnology.

Total cellular proteins were extracted with radioimmunoprecipitation assay (RIPA) buffer (Thermo Scientific, Rockford, IL, USA) containing protease and phosphatase inhibitor cocktails (GenDEPOT, Barker, TX, USA). Cytoplasmic and nuclear lysates were separated using the NE-PER® nuclear and cytoplasmic extraction reagent kit (Thermos Scientific) by following the manufacturer's protocol. Briefly, equal amounts of protein (30-50 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (EMD Millipore, Bedford, MA, USA), which were then blocked in PBST containing 5% skim milk for 1 h and incubated with primary antibodies (1:1000) overnight at 4°C. After three washes with PBST, horseradish peroxidase-conjugated secondary antibodies (1:5000) were added and membranes were incubated for 1 h at room temperature and rinsed. Detection was performed using an enhanced chemiluminescence prime solution (Amersham Bioscience, Buckinghamshire, UK). Bands were visualized using a Fusion Solo 2 M chemiluminescence imaging system (Vilber Lourmat, France).

MMP-related proteins were detected using a human MMP array kit (RayBiotech, USA). The extracted tumor tissues were lysed using a RIPA buffer (GenDEPOT) containing a protease and phosphatase inhibitor cocktail (GenDEPOT). The resulting lysate was applied to the membrane array kit and incubated overnight at 4°C. After several washings, the membrane was incubated with HRP-Streptavidin for 2 h at room temperature, washed and further incubated with the kit detection buffer for 2 min at room temperature. MMP-related protein expression was observed using the ChemiDocTM imager.