Bca kit

Proteins were extracted from samples with a Beyotime protein extraction kit, after which a BCA kit (Qiagen, CA, USA) was used to quantify protein concentrations. Next, 30 μg of protein per sample was separated via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore, Temecula, CA, USA). Blots were subsequently blocked for 2 h with 5% nonfat milk at 37°C followed by staining overnight with antibodies specific for E-cadherin (1:500, Abcam, Cambridge, MA, USA), Notch1 (1:500, Abcam), or GAPDH (1:1000, CST) at 4°C. Blots were then treated for 1 h with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Abcam) at 37°C, after which enhanced chemiluminescence (Amersham Biosciences, IL, USA) was used to visualize protein bands with Image Lab 2.0 (Bio-Rad Laboratories, CA, USA).

Total proteins of cells were extracted, and the protein concentration was
detected in accordance with the instructions of the bicinchoninic acid (BCA) kit
(Qiagen, Wiessberg, Deutch). Then the extracted proteins were run on sodium
dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto
polyvinylidene fluoride membranes. Next, the membranes were blocked with 5%
bovine serum albumin (BSA) at room temperature for 1 h, and then incubated with
following primary antibodies at 4°C overnight: IκBα (1:1000, ab32518,), p-IκBα
(1:10000, ab133462), p65(1:1000, ab32536), p-P65 (1:5000, ab86299) (all
purchased from Abcam, Cambridge, MA, USA). Next, the membranes were incubated
with secondary antibody goat anti-rabbit immunoglobulin G (IgG, 1:2000, ab6721,
Abcam) at room temperature for 1 h. Glyceraldehyde-3-phosphate dehydrogenase
(1:10000, ab181602) was set as an internal reference. The protein bands were
developed using chemiluminescence reagents. The gray value analysis for target
bands was conducted by the Image J software (National Institutes of Health.
Bethesda, Maryland, USA).