Rabbit anti ldha monoclonal antibody

Rabbit anti-EGFR antibody (#2232), rabbit anti-LDHA monoclonal antibody (#3582), rabbit anti-Hexokinase2 monoclonal antibody (#2867), and rabbit anti-β-actin (#4967) antibody were purchased from Cell Signaling Technology (Danvers, MA, USA). Overexpression plasmid containing open reading frame (ORF) of EGFR was obtained from OriGene.com (RC217384). siEGFR, siFGD5-AS1, siHK2, and control siRNA were obtained from Ambion (Austin, TX, USA); shFGD5-AS1 and control shRNA were constructed by Hanbio (Shanghai, China); pre-miR-330-3p, anti-miR-330-3p, and negative control were purchased from GenePharma (Shanghai, China). 5-Fu was purchased from Sigma-Aldrich (F8423, Carlsbad, CA, USA).

Total protein was extracted from the cells and tissues using RIPA lysis buffer according to the manufacturer’s instructions. Proteins were separated using 10% SDS-polyacrylamide gel and then transferred to a polyvinylidene difluoride (PVDF) membrane (GE Healthcare Life Sciences, Chalfont, UK). The PVDF membrane was blocked with phosphate-buffered saline (PBS) buffer containing 5% fat-free milk overnight at 4°C. The membranes were incubated with rabbit anti-LDHA monoclonal antibody (1:1,000; 3582; Cell Signaling Technology, Danvers, MA, USA) and rabbit anti-β-actin monoclonal antibody (1:1,000; 4970; Cell Signaling Technology) at room temperature for 3 h. Subsequently, the membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:1,000; 7074; Cell Signaling Technology) at room temperature for 2 h. The signals on the PVDF membrane were detected using the Enhanced Chemiluminescent Kit (Beyotime) according to the manufacturer’s protocol. The relative protein expression levels were analyzed using Quantity One v4.63 (Bio-Rad, Hercules, CA, USA) and are presented as the density ratio versus β-actin.