Anti ldha

To extract total protein, tissue samples and cells were lysed using ice-cold radioimmunoprecipitation (RIPA) lysis buffer supplemented with protease inhibitors (Solarbio, Beijing, China). The protein concentrations were determined using a protein quantitation kit (Thermo Fisher Scientific, Rockford, IL, USA). NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific) were used for nuclear protein extraction according to the manufacturer’s protocol. Total protein or nuclear protein was then subjected to 10% or 15% SDS-PAGE and transferred onto nitrocellulose membranes (Millipore, Bredford, MA, USA). After the non-specific binding was blocked with 5% nonfat dried milk in TBST for 1 h, the membranes were incubated with primary antibodies at 4°C overnight. The following primary antibodies were used: anti-FAM84B, anti-c-Myc, and anti-LDHA were from Abcam (Cambridge, MA, USA), and anti-Survivin, anti-GAPDH, anti-β-catenin, and anti-H3 were obtained from Cell Signaling Technology (Danvers, MA, USA). After washing with TBST and incubation with corresponding HRP-conjugated secondary antibodies (Beyotime, Shanghai, China), the immunoreaction was detected with enhanced chemiluminescence (Millipore).

Samples of mouse tumor tissue were ground at low temperature, and the tissue seriflux and cells in each group were added to RIPA lysate buffer. After incubation for 40 min on ice, the solution was centrifuged (13400 g for 15 min), and the supernatant was collected. The extracted proteins were quantified using the BCA method (Beyotime, China). Next, a 40 μg aliquot of total protein from each sample was separated by 10% SDS-PAGE, and the protein bands were transferred onto PVDF membranes (Roche, Basel, Switzerland). After blocking, the membranes were incubated with primary antibodies (1: 1000) against the target proteins overnight at 4°C and then subsequently incubated with a secondary antibody (1: 2000) for 2 h. The immunostained protein bands were detected by treatment with ECL solution (Thermo Fisher Scientific, Waltham, MA, USA). The primary antibodies used (anti-caspase 3, anti-HK2, anti-PKM2, and anti-LDHA) and secondary antibody (anti-GAPDH) were purchased from Abcam (Cambridge, MA, USA).

Western blotting was performed on cultured cells or tissue samples after the indicated treatments. Cell lysates were collected using a sodium dodecyl sulfate lysis buffer (Beyotime Biotechnology, Shanghai, China). Equal amounts of total protein (approximately 15 μg for cell samples and 60 μg for tissue samples) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Millipore, Billerica, MA, USA). Membranes were blocked using 5% nonfat powdered milk (Sangon, Shanghai, China) in TBS with Tween-20 (TBST) at about 20°C for 1.5 h, incubated with primary antibodies overnight at 4°C, washed three times with TBST, and then incubated with secondary antibodies for 1 h. The signal intensity of protein bands was visualized using an Enhanced Chemiluminescence Detection Kit (Tanon, Shanghai, China). A semi-quantitative evaluation of protein density was performed using ImageJ (Version 1.5.3). The following antibodies were used at a 1:1000 dilution: anti-GGH (Cat. #138495; Abcam, Cambridge, UK), anti-PKM (Cat. #150377; Abcam), anti-GLUT1 (Cat. #115730; Abcam), anti-LDHA (Cat. #52488; Abcam), anti-β-actin (Cat. #4970, Abcam), and anti-GAPDH (Cat. #2118; Cell Signaling, Danvers, MA, USA).