A19083

Strong RIPA lysis buffers (Meilun, Dalian, China) were used to lye the cells 48 hours after transfection, and total protein was recovered by high-speed centrifugation. The Enhanced BCA Protein Assay Kit (Meilun, Dalian, China) was used to quantify total protein, and 30 g of total protein was segregated using sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The entire proteins were then isolated and put into polyvinylidene fluoride (PVDF). Close for 1 hour at room temperature. The primary antibodies were incubated overnight at 4°C. The following primary antibodies were used: anti-GAPDH antibody (AC002; Abclonal, Wuhan, China; 1:5000 dilution), anti-FoxP3 antibody (ab22510, Abcam; 1:1000 dilution), anti-E2F1 antibody (A2067; Abclonal; 1:1000 dilution), anti-Flag antibody (20543-1-AP; Proteinech, Wuhan, China; 1:1000 dilution), anti-CDH2 antibody (A19083; Abclonal; 1:1000 dilution), anti-PCNA antibody (A12427; Abclonal; 1:1000 dilution), and anti-Snail antibody (A11794; Abclonal; 1:1000 dilution). A goat anti-mouse IgG(H+L) secondary antibody conjugated with horseradish peroxidase (HRP) was then added (31432; Invitrogen, Carlsbad, CA, USA; 1:2000 dilution) to the membranes, followed by washing with wash buffer and incubation with a goat anti-mouse or a goat anti-rabbit IgG(H+L) secondary antibody.

Cells were lysed by PMSF added lysate (ST506 and P0013, Beyotime, China). Protein concentration was measured by a BCA detection kit (P0011, Beyotime, China). Then, the protein was subjected to SDS-polyacrylamide gel electrophoresis (P0015, Beyotime, China) and transferred to polyvinylidene fluoride (PVDF) membranes (IPVH00010, Millipore, USA). After blocked with 5% nonfat milk (YiLi, China) for 1 h at room temperature, the membrane was incubated with primary antibody overnight at 4 °C, followed by horseradish peroxidase (HRP)-conjugated goat anti-rabbit (A0208, Beyotime, China) or anti-mouse (A0216, Beyotime, China) antibody at 1:5000 dilution for 45 min. The blots were incubated in ECL substrate (P0018, Beyotime, China), and images were analyzed using the Gel-Pro-Analyzer software.
Antibody information: NREP (1:500, PA5-68426, Thermofisher, USA), HIF-1α (1:500, AF1009, Affinity, China), E-cadherin (1:1000, A20798, Abclonal, China), N-cadherin (1:500, A19083, Abclonal, China), SLUG (1:1000, A1057, Abclonal, China), MMP9 (1:500, A0289, Abclonal, China), PKM2 (1:500, A13905, Abclonal, China), GLUT1 (1:1000, AF5462, Affinity, China), HK-2 (1:500, A20829, Abclonal, China), and LDHA (1:500, A1146, Abclonal, China).