Ab131030

Proteins were extracted using RIPA lysis buffer (Beyotime, Beijing, China). Extractive proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then shifted onto polyvinylidene fluoride membranes. The antibodies used in this study included cleaved-caspase3 (C-caspase3) (1 : 1000, ab2302, Abcam, Cambridge, MA, USA), matrix metallopeptidase 2 (MMP2) (1 : 5000; ab92536, Abcam), MMP9 (1 : 2000, ab38898, Abcam), LMO4 (1 : 5000, ab131030, Abcam), proliferating cell nuclear antigen (PCNA) (1 : 5000, ab29, Abcam), GAPDH (1 : 10000, ab181602, Abcam) primary antibodies (overnight, 4°C), and HRP-conjugated secondary antibody (1 : 3000, Sangon, Shanghai, China) (1 h, 37°C). The same experiment was repeated three times.

Whole-cell lysate was prepared using ice-cold RIPA buffer with protease inhibitors. Then, protein samples were resolved via 10% SDS-PAGE electrophoresis. Nitrocellulose membranes were employed to transfer the proteins. Next, membranes were incubated with the following primary antibodies: rabbit anti-LMO4 monoclonal antibody (Abcam, ab131030) and anti-GAPDH polyclonal antibody (Abcam, ab9485). Subsequently, incubation with goat anti-rabbit IgG (Abcam, ab205718) or goat anti-mice IgG (Abcam, ab205719) was conducted. Finally, immunoreactive bands were observed using an ECL reagent (Pierce; Thermo Fisher Scientific, Inc.).

Total protein was extracted from cells or tissues using high-performance RIPA lysis buffer (R0010, Solarbio), with the concentration determined using a bicinchoninic acid (BCA) assay kit (Yeasen, Shanghai, China). The proteins from each sample were then separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA) via the wet-transfer method. The membrane was blocked with 5% bovine serum albumin for 1 h at room temperature and incubated with primary antibodies against FOXM1 (ab180710, 1:1000, Abcam, Cambridge, UK), LMO4 (ab131030, 1:1000, Abcam), AKT (ab8805, 1:500, Abcam), PI3K (ab40776, 1:1000, Abcam), or phosphorylated (p)-PI3K (ab182651, 1:1000, Abcam), followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (ab205718, 1:10000, Abcam) at room temperature for 1 h. Development was performed using VILBER FUSION FX5 (Vilber Lourmat, France). The protein bands were quantified using ImageJ 1.48 (National Institute of Health, Bethesda, MD, USA), and normalized to GAPDH. Each experiment was performed in triplicate.