Anti e cadherin ab40772

Cells were washed three times with PBS, lysed with RIPA buffer on ice for half an hour and centrifuged at 12,000 g/s for 20 min to collect the supernatant. We used a BCA kit (Boster Biological Technology) to detect the protein concentration. Then, the proteins (10 µL/lane) were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk, probed with primary antibodies (1:1000) and stained with horseradish peroxidase (HRP)-linked secondary antibodies (1:15,000, Promega). Then, the blots were visualized using an ECL kit (Tiangen). The antibodies used in this study were as follows: anti-PTEN (Proteintech, 22034-1-AP), anti-β-actin (Proteintech, 20536-1-AP), and anti-β-Catenin (Proteintech, 66379-1-lg). Anti-p-mTOR (5536), anti-mTOR (2983), anti-P65 (8242), anti-IKB (4814), anti-p-ERK1/2 (4370), anti-ERK1/2 (5013), anti-p-STAT3 (9131), anti-STAT3 (9132), anti-CDK2 (18048), anti-CyclinB1 (12231), anti-P21 (2947), anti-cleaved caspase-3 (9664), anti-cleaved caspase-9 (20750), anti-p-GSK-3β (5558) and anti-GSK-3β (12456) were purchased from Cell Signaling Technology [CST, Danvers, MA (20750)]. Anti-p-JAK2 (ab32101), anti-JAK2 (ab108596), anti-vimentin (ab92547), anti-N-cadherin (ab18203) and anti-E-cadherin (ab40772) were purchased from Abcam (MA, United States).

At 24 h post transfection, the cells were collected and seeded onto a six-well plate. After 48 h, the cells were lysed and sonicated. Then, protein concentrations of lysates were measured using a Protein Assay Kit (Bio-Rad, Hercules, CA). The lysates were re-suspended in Laemmli buffer, denatured for 5 min at 98°C, and separated by Tris-glycine denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins were blotted onto polyvinylidene fluoride membranes, blocked in 5% milk, and incubated overnight with the following primary antibodies: for endogenous protein detection, anti-E-cadherin (ab40772; Abcam, Cambridge, United Kingdom) or anti-α-tubulin (ab11304; Abcam); for MS2-22sTag protein detection, anti-HA (ab49969; Abcam) at a 1:1,000 (ab40772 and ab11304) or 1:2,000 (ab49969) dilution ratio in Can Get Signal Solution 1 (Toyobo) at 4°C. Subsequently, the proteins were incubated with the corresponding horseradish peroxidase–conjugated secondary antibodies (Thermo Fisher Scientific) at a 1:250 dilution ratio in Can Get Signal Solution 2 (Toyobo) for 1 h at room temperature. Chemiluminescent signals were generated using a SuperSignal West Pico Plus Chemiluminescent Substrate (Thermo Fisher Scientific) and captured on X-ray films (Fujifilm, Tokyo, Japan). The films were scanned, and signal intensities were quantified using ImageJ software.^*