Anti β actin monoclonal antibody

Aliquots (120 mg) of total proteins from each tissue were separated on 10% SDS-acrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane in blotting buffer (20 mmol/L Tris-base, 150 mmol/L glycine and 20 % methanol). The glycoprotein were then probed using lectin detection with biotinylated ABL (Vectorlabs), anti-β-actin monoclonal antibody (Abcam), and anti-microtubule-associated protein 6 monoclonal antibody (STOP (175): sc-53513, anti-MAP6, Santa Cruz Biotechnology, Santa Cruz, CA). Protein bands were visualized using an enhanced chemiluminescence system (GE Healthcare Bio-Sciences Corp.).

The expression of both RRM2 and equilibrative nucleoside transporter-1 (hENT1) markers were assessed by Western blotting. Cells were treated with drug-loaded nanoparticles or controls for 24 h, and washed with PBS. Total protein was harvested and quantified using the bicinchoninic acid protein assay kit (Thermo Fisher Scientific). Protein samples were prepared at a concentration of 50 μg protein/20 μL. Samples in loading buffer were heated at 75 °C for 10 min, loaded into wells of 15% acrylamide gels (Thermo Fisher Scientific) with a protein ladder, and run on SDS-PAGE at 100 V. The proteins were subsequently transferred onto nitrocellulose membranes (Thermo Fisher Scientific) for 1 h and blocked at 4 °C overnight. Blots were treated with an anti-RRM2 antibody (1:1000, Abcam) or an anti-hENT1 antibody (1:1000, Abcam) in blocking buffer for 1 h at room temperature on a shaker, while an anti-β-actin monoclonal antibody was a loading control (1:1000, Abcam). After washing with Tris Buffered Saline with Tween (TBST, Thermo Fisher Scientific) 4 times, the blots were incubated with secondary antibodies (1:5000, Abcam) in the dark for 1 h. After washing 4 times and drying, the membranes were scanned with the Odyssey imaging system (LI-COR) (LI-COR Biosciences, Lincoln, Dearborn, MI, USA).