Anti hexokinase 2 anti hk2

Cells and tumor tissues were lysed using RIPA buffer (Beyotime). Lysates were mixed with loading buffer (Thermo Fisher), which was then boiled in boiling water. Then samples were loaded on 12% bis–tris-acrylamide gel (Thermo Fisher) and then electrotransferred onto polyvinylidene fluoride (Millipore, Bradford, MA, USA). After immersed in 5% nonfat milk diluted with tris buffered saline tween (Millipore), membranes were incubated with anti-recombinant glucose transporter 1 (anti-GLUT1) (1:200; Abcam), anti-hexokinase 2 (anti-HK2) (1:1000; Abcam), anti-YWHAZ (1:1000; Abcam) and anti-β-actin (1:1000; Abcam). Secondary antibody labeled with horseradish peroxidase (1:10,000; Abcam) was subsequently used to incubate membranes. Protein bands were visualized using RapidStep ECL Reagent (Millipore). β-actin acted as a control.

HCT116 and LoVo cells were lysed. The sample was loaded into 12% SDS-PAGE. After that, proteins bands were transferred onto nitrocellulose membranes. Following bands were blocked in 5% skim milk. Then, membranes were incubated with primary antibodies. Following, a secondary antibody labeled with horseradish peroxidase was utilized to incubate membranes. Results were visualized by enhanced chemiluminescence (Pierce, Rockford, IL, USA). GAPDH was chosen as a control. The primary antibodies were anti-hexokinase 2 (anti-HK2) (1:1000; Abcam), anti-MYO6 (1:500; Abcam), anti-proliferating cell nuclear antigen (anti-PCNA) (1:1000; Abcam), and anti-GAPDH (1:2500; Abcam).