A1146

Western blot was performed as previously described.²⁹ Proteins were probed using primary antibodies against HK2 (A0994, 1:1000; ABclonal), LDHA (A1146, 1:1000; ABclonal), PKM2 (A19102, 1:1000; ABclonal), β‐actin (ab133626, 1:5000; Abcam).

Cell lysates were prepared in RIPA lysis buffer with the addition of a mammalian protease inhibitor cocktail (Sigma) and phosphatase inhibitor. Protein concentration was measured by BCA protein assay kit. Equal amounts of cell lysates were electrophoresed on SDS-polyacrylamide gels and transferred by electroblotting onto a polyvinylidene fluoride (PVDF) membrane. The following primary antibodies were used: mouse SHP1 (1:1000; abs158291, ABsin), and rabbit PKM2 (1:1000; 4053, CST Technology), p-PKM2 (1:1000; 3827, CST Technology), LDHB (1:1000; A5131, ABclonal), LDHA (1:1000; A1146, ABclonal). Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:10000; ABclonal Technology). After one-hour incubation, membranes were washed three times with TBST, and then visualized by immunoblotting.

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).