Millipore ecl

The cells were washed three times with cold PBS buffer, lysed with RIPA Buffer (Beyotime, China) and placed on ice for 30 min. Proteins were separated on SDS-PAGE and subsequently electro transferred to a PVDF membrane (Millipore, United States). The membrane was blocked with 5% BSA in Tris-buffered saline and Tween 20 (10 mM Tris, pH 7.5, 140 mM NaCl, 0.05% Tween-20) for 2 h at room temperature. The membrane was incubated with specific primary antibodies (Rabbit polyclonal anti-RANK, 1:1000 dilution; Rabbit monoclonal anti-Traf6, 1:2000 dilution; Rabbit polyclonal anti-Fra-1, 1:1500 dilution; Mouse monoclonal anti-c-src, 1:1000 dilution, Abcam, Cambridge, MA, United States) followed by horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies. Millipore ECL (Millipore, United States) was used for antibody detection according to manufacturer’s instructions.

The breast cancer cells were gently rinsed four times with 1 × PBS and harvested in cell lysis buffer containing 1 mM phenylmethanesulfonyl fluoride (PMSF, cat. no. P0100, Solarbio). The cells were lysed on ice for 10 min. The whole cell lysate was centrifuged at 1,2000 g for 10 min at 4 °C, and the supernatant was collected for subsequent applications. Equal amounts of protein were isolated by 12% SDS-PAGE, and the proteins were transferred to polyvinylidene fluoride (PVDF, cat. no. ISEQ00010, Merck Millipore, Co., Ltd.) membranes by membrane electrophoresis for 2 h. The membranes were then sealed with TBS‑5% Tween‑20 (TBST) containing 5% skim milk at room temperature for 2 h to prevent binding of nonspecific antibodies. The dilutions of various primary antibodies were applied, followed by overnight incubation at 4 °C at 1:1000 concentration as recommended by the supplier. Then, the PVDF membranes were washed three times with TBST (10 mM Tris, 10 mM NaCl, 0.1% Tween—20), and incubated for 2 h with the secondary antibody at a concentration of 1:10,000. The samples were tested with Millipore ECL (WBKLS0100, Millipore). An antibody against actin was used to control protein loading and transfer. ImageJ version 6 (National Institutes of Health) was used to measure the strip density.

Total protein was extracted according to the relevant instructions, and BCA method was used for protein quantitation. The supernatant from the above lysate (containing about 80 μg of protein) was subjected to 12% SDS-PAGE and transferred onto a nitrocellulose (NC) membrane. The membranes were probed with primary antibodies against ZO-1, E-cadherin, TLR-4, NF-κB, and β-actin followed by incubation with secondary antibodies conjugated with horseradish peroxidase (HRP, Thermo Fisher, Waltham, MA, USA). Millipore ECL (Millipore, Massachusetts, USA) was used for chemiluminescence, film exposure, and fixation, and grayscale value was analyzed by using Image Lab software (BioRad, Hercules, CA, USA).