Rabbit anti human gapdh

Protein expression levels of DYNLRB2 in transfected cells were confirmed by Western blot analysis. A549 transfected cells were harvested 48 h after transfection and extracted by lysis buffer with 0.01% protease inhibitor and phenylmethylsulphonyl fluoride. The cells were centrifuged at 12,000 rpm at 4 °C for 5 min to remove the supernatant. The protein concentration was measured using the BCA Protein Quantitation Kit (Thermo Fisher, MA, USA). Each 20 μL sample was added to 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis. The polyvinylidene difluoride membranes were blocked for 2 h with 5% non-fat milk in Tris-buffered saline containing 0.05% Tween-20 (TBST) at room temperature. The membranes were further incubated with primary antibodies targeting rabbit anti-human DYNLRB2 (Sigma-Aldrich, St. Louis, MO, USA) (1:1,000) and rabbit anti-human GAPDH (Sigma-Aldrich, St. Louis, MO, USA) (1:1,000) at 4 °C overnight, then washed 5 times with TBST. The membranes were further incubated with horseradish peroxidase-conjugated secondary antibody (Bio-Rad, Hercules, CA, USA) (1:50,000) diluted in TBST. After washing, the membranes were reacted via enhanced chemiluminescence (ECL) solution (Bio-Rad, Hercules, CA, USA). Finally, the gel band intensity was analyzed by BandScan 5.0 software for protein concentration.