Pierce ecl 2 western blotting substrate

For immunoblot analysis, cells were lysed with Laemmli buffer (125 mm Tris-HCl [pH 6.8], 4% SDS, 20% glycerol, 100 mm dithiothreitol [DTT], 0.02% bromphenol blue), boiled for 5 min, sonicated and protein content was resolved by SDS-polyacrylamide gel electrophoresis. Proteins were then transferred on PVDF membranes (GE Healthcare) and detected after incubation with specific antibodies using Pierce ECL 2 Western blotting Substrate (Fisher Scientific).

Cells were homogenized in RIPA buffer supplemented with protease inhibitors (Complete tablets, Roche Diagnostics, Cat# 11836153001), 1 mM AEBSF (ThermoFisher Scientific, Cat# 50-213-115), and 1 mM Na₃VO₄ (Sigma, Cat# S6508-10G). Antibodies for western analyses included: phospho-RON (R&D Systems, Cat# AF1947), RON-β (C-20) (Santa Cruz Biotechnology, Cat# SC-322), β-CATENIN (Cell Signaling Technology, Cat# 9582S and BD Biosciences, Cat# 610154), FLAG (Sigma, Cat# F1804), HGFL (T-19) (Santa Cruz Biotechnology, Cat# SC-6090), phospho-NF-κB p65 (Cell Signaling Technology, Cat# 3033S), NF-κB p65 (Cell Signaling Technology, Cat# 8242S), TUBULIN (Santa Cruz Biotechnology, Cat# SC-5286), and C4-ACTIN (Cincinnati Children's Hospital Medical Center). Peroxidase-conjugated secondary antibodies were applied and membranes were developed using Pierce ECL2 Western Blotting substrate (ThermoFisher Scientific, Cat# PI80196×3).

Livers were lysed with RIPA buffer (9806; Cell Signaling) containing 1 mM phenylmethylsulfonyl fluoride (8553S; Cell Signaling). The protein concentration of each sample was determined using a bicinchoninic acid (BCA) assay reagent (Vigorous Biotechnology) according to the manufacturer’s recommendations. An equal amount of each protein sample was electrophoresed on a 10% acrylamide gel and the bands were then transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad). The membrane was blocked with 5% non-fat dry milk for 3 h and incubated with GFP antibody (#2555; Cell Signaling) and internal control β-actin antibody (ab8227; Abcam) overnight at 4°. The PVDF membrane was then washed three times for 30 min in 0.1% Tween-20 in Tris-buffered saline (TBST) and incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit IgG (Zhongshan). After washing for 30 min with three changes of TBST, the membrane was treated with the Pierce ECL 2 western blotting Substrate (Thermo Scientific).

Cells were lysed in RIPA buffer containing phosphatase inhibitor cocktail (Sigma) and protease inhibitor cocktail (Sigma) on ice for 15 min, and protein content was quantified using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Ten micrograms of protein per sample was resolved by electrophoresis and transferred to PVDF membranes. Membranes were blocked for 1 h at room temperature with 5% milk in Tris-Buffered Saline containing 0.1% Tween-20 (TBS-t; Sigma) and incubated overnight with either anti-TCOF1 (1:1,000; Abnova) or anti-beta actin (1:10,000; Sigma). Membranes were washed with TBS-t and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. Membranes were washed and developed using the Pierce ECL2 western blotting substrate (Thermo Fisher Scientific).

Whole cell lysates were prepared in Pierce™ RIPA Lysis and Extraction Buffer (Thermo Scientific, Rockford, IL, USA, PI89900) and Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific, Rockford, IL, USA, PI78441). Proteins were separated by SDS page, transferred to PVDF membranes (Novex, Littleton, CO, USA, LC2002), and stained with Ponceau S. The membranes were blocked in 5% nonfat dry milk, followed by overnight incubation at 4 degrees with primary antibodies against γH2AX (Cell Signaling Technology, Beverly, MA, USA, 9718S), pS516 CHK2 (Cell Signaling Technology, Beverly, MA, USA, 2669S), pT68 CHK2 (Cell Signaling Technology, Beverly, MA, USA, 2661S), or pT284 XRCC1 (Invitrogen, Carlsbad, CA, USA, PA5-64861). Following primary antibody incubation, the membranes were incubated with an HRP-conjugated secondary antibody and imaged using the Pierce™ ECL 2 Western Blotting Substrate (Thermo Scientific, Rockford, IL, USA, PI80196) according to the manufacturer’s protocol. The membranes were probed with either α-tubulin (Calbiochem, Burlington, MA, USA, CP06) or histone H3 (Cell Signaling Technology, Beverly, MA, USA, 9715S) as a loading control.