Bradford method

Whole cell lysates were prepared from cells that were untreated or treated with glucose and NTS or MSM by incubating them on ice with a radioimmunoprecipitation lysis buffer (20–188; EMD Millipore) containing phosphatase and protease inhibitors to isolate protein. The protein concentrations were measured using the Bradford method (Thermo Fisher Scientific, Inc., Waltham, MA). Equal amounts of proteins (150 μg/well) were resolved by 10–15% SDS-PAGE. Then, the separated proteins were transferred onto nitrocellulose membranes. The blots were blocked for 1 h with 5% skim milk (BD Biosciences, CA; 90002-594) in TBS-T buffer (20 mM Tris–HCl (Sigma-Aldrich; Merck KGaA, St. Louis, MO; 10708976001), pH 7.6, 137 mM NaCl (Formedium, Norfolk, UK; NAC03), 0.1X Tween 20 (Scientific Sales, Inc. Oak Ridge, TN; 0777)) at room temperature. The membranes were then incubated with primary antibodies diluted in 5% skim milk overnight at 4 °C in a shaker. The membranes were then washed with TBS-T and incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies at room temperature. Detection was conducted with a Femto Clean Enhanced Chemiluminescence Solution Kit (GenDEPOT; 77449; Katy TX), and images were acquired using a LAS-4000 imaging device (Fujifilm, Tokyo, Japan).

Human liver samples were homogenized with 300 μl lysis buffer containing 10 mM HEPES pH 7.9, 137 mM NaCl, 10% glycerol, 1% NP-40, 1mM PMSF supplemented with protease inhibitor cocktail (Roche, South San Francisco, CA, USA). Total protein concentrations were measured using Bradford method (Thermofisher Scientific, California, USA). MCF7 whole cell lysates prepared with RIPA lysis buffer (Millipore Sigma) were used as a positive control. Capillary Western blot analyses were performed using the Protein Simple Jess system (Biotechne, California, USA) according to manufacturer’s protocol. Briefly, tissue or cell lysates were diluted with 0.1 × sample buffer to concentration of 1 mg/ml. Then 4 parts of diluted samples were combined with 1 part 5 × Fluorescent Master Mix (containing 5 × sample buffer, 5 × fluorescent standard and 200 mM DTT) and heated at 95°C for 5 min. Then the denatured samples, blocking reagent, mouse anti- ESR1 antibodies (D-12 and F-10, at 1:10 dilution, Santa Cruz, California USA), HRP-conjugated anti-mouse secondary antibody (1:20) and chemiluminescent substrate (Biotechne, California, USA) were dispensed into designated wells in an assay plate. A biotinylated ladder provided molecular weight standard for each assay. After plate loading, the separation, electrophoresis and immunodetection steps take place in the fully automated capillary system.

Whole cell and nuclear lysates were prepared as described previously [9 (link)]. The Bradford method (Thermo) were performed to detect the protein concentrations, Approximately 30 μg of protein was loaded onto gels for sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a nitrocellulose membrane (Bio-Rad), which were incubated with primary antibodies (Keap1, 1:2000, ab139729, abcam, UK; Nrf2, 1:3000, sc-365,949, Santa Cruz, CA; HO-1, 1:1000, sc-103,492, Santa Cruz, CA; GAPDH, 1:6000, sc-20,358, Santa Cruz, CA) and visualized by an enhanced chemiluminescence kit (Roche).

NHBE cell lysates were analyzed for Krüppel-like factors 4 and 5 (KLF4 and KLF5) and phospho-KLF4 and phosphor-KLF5 by Western blot. Protein concentration was determined by Bradford method (Thermo Scientific, Rockford, IL) and 10 μg was loaded per lane for SDS-polyacrylamide gel electrophoresis (8%). Blots of protein bands were probed with primary (KLF4 (rb, 1 : 500 dilution, EMD Millipore, Billerica, MA); p-KLF4 (Ser245) (rb, 1 : 500, Abgent, San Diego, CA); KLF5 (rb, 1 : 500, AVIVA System Biology, San Diego, CA); and p-KLF5 (Ser311) (rb, 1 : 500, Bioss Inc., Woburn, MA; beta-actin) (mo, 1 : 1000, Santa Cruz, Dallas, TX)) and appropriate secondary antibodies and intensity of the bands was measured with ImageJ 1.42 software.

Protein assay in homogenates prepared as described above was performed spectrophotometrically using the Bradford method (Thermoscientific; 23200). Measurements in specimens diluted 1:20 were performed at 595nm, and the protein level in a specimen was determined based on a standard.

Whole cell lysates were prepared from untreated or TA-treated NCCIT cells by incubating them on ice with radio immunoprecipitation lysis buffer (20-188; EMD Millipore) containing phosphatase and protease inhibitors to isolate protein. The protein concentrations were measured via the Bradford method (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Equal amounts of protein (100 μg/well) were resolved with 10% SDS-PAGE. Then, the separated proteins were transferred onto nitrocellulose membranes. The blots were blocked for 1 h with 5% skim milk (BD Biosciences, San Jose, CA, USA; 90002-594) in TBS-T buffer (20 mM Tris-HCl (Sigma-Aldrich; Merck KGaA, St. Louis, MO, USA; 10708976001), pH 7.6, 137 mM NaCl (Formedium, Norfolk, UK; NAC03), 0.1X Tween 20 (Scientific Sales, Inc. Oak Ridge, TN, USA; 0777)). The membranes were then incubated overnight at 4 °C in a shaker with primary antibodies diluted in 5% bovine serum albumin (EMD Millipore). The membranes were then washed with TBS-T and incubated for 1 h at room temperature with Horseradish peroxidase (HRP)-conjugated secondary antibodies. Detection was performed with a Femto Clean Enhanced Chemiluminescence Solution Kit (GenDEPOT; 77449; Katy TX) and a LAS-4000 imaging device (Fujifilm, Tokyo, Japan).