Atad2

The proteins were extracted by ice cracking. SDS-PAGE was used to separate the proteins. Subsequently, the target proteins were transferred onto a polyvinylidene difluoride membrane at low temperatures, followed by treatment with 5% milk for 50 min. Immediately afterward, the membranes were incubated overnight at 4°C with a primary antibody (ATAD2, 1 : 1000, #78568, Cell Signaling; VEGFA, 1 : 10000, ab52917, Abcam; and GAPDH, 1 : 5000, abs132004, Absin). In the following day, the membranes were removed and subjected to the process of PBST washing, treatment with a secondary antibody (anti-rabbit IgG, HRP-linked antibody, 1 : 5000, #7074 Cell Signaling), PBST rinsing, and, finally, exposure to the chromogenic apparatus by dropwise addition of the chromogenic solution for color development.

The cells were lysed in cell lysis buffer (Beyotime Biotechnology, China) to obtain the protein, and the concentration of the protein was measured with a BCA Kit (Beyotime Biotechnology, China). Protein (30 μg per lane) was added to a polyacrylamide gel and transferred to a PVDF membrane after separation by electrophoresis. Subsequently, the membrane was blocked with TBST diluted 5% skim milk for 1 hr at room temperature. Primary antibodies against ATAD2 (1:1000; Cell Signaling Technology), E-cadherin (1:1000; Cell Signaling Technology), N-cadherin (1:1000; Cell Signaling Technology) and Snail (1:1000; Cell Signaling Technology) were used for binding protein specifically at 4°C overnight. After washing three times for 10 min in TBST, membranes were incubated with an HRP-labeled goat anti-rabbit IgG (1:10000, Proteintech, Wuhan, China) for 1 hr at 37°C. The WesternBright Sirius Chemiluminescent Detection Kit was used for the visualization of bands on PVDF membranes. GAPDH (1:10000, Bioprimacy) was used as an internal reference protein. The experiments were repeated three times.

Tissues or cells were lysed with T-PER Tissue Protein Extraction Reagent (Thermo Fisher Scientific Inc., Rockford, IL) for 15 min on ice, and then sonicated with power 1 for 2 seconds. Supernatants were collected for measurement of protein concentration. Protein lysates (10 μg) were suspended in loading buffer and separated on 10% SDS-polyacrylamide gels and then transferred onto nitrocellulose membranes for incubation with primary antibodies overnight at 4°C. This was followed by incubation with HRP-conjugated secondary antibodies. Immuno-complexes were detected by Supersignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific Inc., Rockford, IL) according to the manufacturer's protocol. Primary antibodies were: ATAD2, c-PARP, p-p53, Puma, Bax, Bad, Bak, Bim, and Bcl-xL (Cell Signaling Technology Inc., Danvers, MA); p38, MKK3/6, p-MKK3/6, GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA) and Actin (Sigma-Aldrich, St. Louis, MO). Secondary antibodies were: goat anti-rabbit and goat anti-mouse (Santa Cruz Biotechnology, Santa Cruz, CA).