Amersham ecl prime western blotting reagent

The total protein and nuclear protein were extracted with Cell Lysis Buffer (Cell Signaling) and NE-PER Nuclear Protein Extraction Kit (Thermo Scientific). The collected proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were incubated overnight at 4 °C with primary antibodies of TRIB2 (1:1000, #13533), CEBPA (1:1000, #2295), PARP (1:1000, #9542), cleaved-PARP (1:1000, #5625), cleaved-Caspase 3 (1:1000, #9664), Cofilin (1:1000, #3312), and GAPDH (1:5000, #2118), all from Cell Signaling. After washing, the membranes were incubated for 2 hours at room temperature with anti-rabbit horseradish peroxidase-coupled secondary antibody (1:5000, GE Healthcare). The membranes were then developed with Amersham ECL Prime Western Blotting Reagent (GE Healthcare), and illuminated on premium autoradiography films (Denville Scientific).

E-cadherin antibody was purchased from BD Biosciences. Plakoglobin and Paxillin (PXN) antibodies were purchased from Millipore. Antibodies against Snail1, mitogen-activated protein kinase (ERK1/2), phospho-p44/42 ERK1/2, SRC, phospho-SRC (pY416), focal adhesion kinase (FAK), phospho-PXN (pY118), phospho-Akt (pS473), β-actin and HRP-conjugated secondary antibodies were purchased from Cell Signaling Technology. Phospho-FAK (pY861) antibody was purchased from Thermo Scientific.
Cells are grown on 6-well plates and treated with compounds or DMSO as control for 48 h. Cells were rinsed with PBS and lysed with RIPA buffer supplemented with protease / phosphatase inhibitor cocktail (Promega). Protein concentration was quantified and equalized protein loads were resolved with Bio-Rad SDS-PAGE system using 8% to 12% polyacrylamide gels and transferred to PVDF membranes. Immunoblotting were performed with the antibodies listed above and bound antibodies were detected by chemiluminescence using Amersham ECL Prime Western Blotting reagent (GE Healthcare).

A 50 µg of S. mutans protein lysate was separated via 14% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and transferred to a nitrocellulose blotting membrane with 0.45 μm pore size (transfer buffer: 25 mM Tris, 200 mM glycine, and 20% [vol/vol] methanol) for 75 min at 100 V. The membrane was blocked in PBS containing 3% (wt/vol) skim milk (Sigma) for 30 min at room temperature and subsequently incubated with primary antibodies overnight at 4 °C. Monoclonal anti-FLAG M2 primary antibody (Sigma # F3165) was used at a 1:2000 dilution, while the HA Epitope Tag primary antibody (ThermoFisher # 26183) was used at a 1:4000 dilution in PBS with 3% (wt/vol) skim milk. The membrane was washed with PBS, washed twice with tris-buffered saline containing 0.1% (vol/vol) Tween-20 (TBST), followed by a final wash in PBS. Afterward, the membrane was incubated with anti-IgG secondary antibody (Sigma # 12-349) at a 1:2000 dilution in TBST for 1–2 h at room temperature. Washing steps were repeated as described above. For signal detection, the membrane was incubated with Amersham ECL Prime Western Blotting Reagent (GE Healthcare) and imaged using an ImageQuant LAS 4000 (GE Healthcare). All blots presented as part of the same series were derived from the same experiment and were processed in parallel. Original blots are provided in Supplementary Information.