Anti pgk1

The IHC staining was used to examine the expression levels of c-Myc and glycolytic enzymes. The paraffin-embedded sections were incubated in a pressure cooker containing antigen retrieval buffer (pH 6.0) (Solarbio; G1202). Next, the slides were uniformly covered with 3% bovine serum albumin (Solarbio; A8020), and probed with the following primary antibodies prepared in a wet box at 4 °C overnight: anti-c-Myc (Servicebio; GB13076), anti-GLUT1 (Proteintech, 21,829–1-AP), anti-ENO1 (Proteintech, 11,204–1-AP), anti-PGK1 (Proteintech, 17,811–1-AP), anti-ALDOA (Proteintech, 11,217–1-AP), anti-HK1 (Proteintech, 19,662–1-AP), and anti-LDHA (Proteintech, 19,987–1-AP) antibodies. Next, the horseradish peroxidase (HRP)-labeled secondary antibody was used for 50 min. Then, the sections were then counterstained with hematoxylin.
The stained sections were scanned using Pannoramic MIDI and analyzed using Quant Center, which automatically identified all the strongly positive, moderately positive, weakly positive, and negative areas in the tissue sections. The H-scores were calculated as ∑ (percentage of intensity × intensity). The staining intensity was divided into three categories—strong, moderate, and weak—and the corresponding score was 3, 2, and 1, respectively.

We performed immunohistochemical staining and western blotting using methods that have been described previously [24 (link)]. Anti-YAP (1:100, immunohistochemistry), Anti–phosphorylated-YAP (p-YAP) (Ser127) (1:1000, western blot), and anti–p-LATS1 (Ser909) (1:1000, western blot) were from Cell Signaling Technology (Shanghai, China). Anti-YAP (1:1000, western blot), anti-LDHA (1:1000, western blot), anti-PKM2 (1:1000, western blot), anti-Glut1 (1:1000, western blotting), anti-PGK1 (1:1000, western blot), anti–large tumor suppressor kinase 1 (LATS1, 1:1000, western blotting), and anti–HIF-1α (1:1000, western blot) rabbit monoclonal antibodies were from Proteintech (Wuhan, China). Anti–matrix metalloproteinase 2 (MMP2, 1:1000, western blot), anti-MMP9 (1:1000, western blot), horseradish peroxidase–linked secondary antibody (1:5000, western blot), and anti–β-actin (1:3000, western blot) were from Abbkine (Wuhan, China). The protein bands were analyzed with Image J software (National Institutes of Health, Bethesda, MD, USA).