Ab236422

Cells were lysed with RIPA buffer (Solarbio). Subsequently, proteins were separated by polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking with 5% skim milk for 1 h, the membranes were incubated with primary antibodies against hexokinase II (HK2; 1:5000, ab227198, Abcam), lactate dehydrogenase A (LDHA; 1:1000, ab125683, Abcam), TRIM44 (1:2000, ab236422, Abcam) or β-actin (1:2000, ab8227, Abcam). Next, the membranes were washed with TBST buffer 4 times. Then, the membranes were probed with secondary antibody (1:20000, ab205718, Abcam) for 2 h at room temperature and then washed with TBST buffer 4 times. All antibodies were diluted with TBST buffer. The protein bands were visualized using the enhanced chemiluminescence system (Qiagen).

Protein was extracted from tissues and cells for analysis. The samples were boiled at 100 °C for 5 min. Equal amounts of protein were separated by SDS-PAGE followed by transfer to PVDF membranes (Millipore, USA). The membranes were incubated with primary antibodies overnight at 4 °C. The following primary antibodies: mouse anti-human TRIM44 (ab236422; 1:1,000 dilution; Abcam, Cambridge, UK), rabbit anti-human CCND3 (ab112034, 1:1,000 dilution; Abcam, Cambridge, UK), rabbit anti‐human FOXM1 (ab245309, 1:1,000 dilution, Abcam, Cambridge, UK), rabbit anti-human EZH2 (#4905,1:100 dilution, Cell Signaling Technology), rabbit anti-human CCNE2 (ab226388, 1:1,000 dilution; Abcam, Cambridge, UK), rabbit anti-human BIRC5 (PAB18224, 1:1,000 dilution; Abnova, China), and mouse anti-GADPH (AG019, 1:1,000 dilution, Beyotime) as internal controls. Immunoreactive protein bands were detected using an ECL detection system (Cell Signaling Technology, USA).